Compositions and methods for modulating apolipoprotein c-iii expression

ABSTRACT

Provided herein are oligomeric compounds with conjugate groups targeting apolipoprotein C-III (ApoCIII). In certain embodiments, the ApoCIII targeting oligomeric compounds are conjugated to N-Acetylgalactosamine. Also disclosed herein are conjugated oligomeric compounds targeting ApoCIII for use in decreasing ApoCIII to treat, prevent, or ameliorate diseases, disorders or conditions related to ApoCIII. Certain diseases, disorders or conditions related to ApoCIII include inflammatory, cardiovascular and/or metabolic diseases, disorders or conditions. The conjugated oligomeric compounds disclosed herein can be used to treat such diseases, disorders or conditions in an individual in need thereof.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0249USC3SEQ_ST25.txt, created on Feb. 5, 2018, which is 68 Kb insize. The information in the electronic format of the sequence listingis incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The principle behind antisense technology is that an antisense compoundhybridizes to a target nucleic acid and modulates the amount, activity,and/or function of the target nucleic acid. For example in certaininstances, antisense compounds result in altered transcription ortranslation of a target. Such modulation of expression can be achievedby, for example, target mRNA degradation or occupancy-based inhibition.An example of modulation of RNA target function by degradation is RNaseH-based degradation of the target RNA upon hybridization with a DNA-likeantisense compound. Another example of modulation of gene expression bytarget degradation is RNA interference (RNAi). RNAi refers toantisense-mediated gene silencing through a mechanism that utilizes theRNA-induced silencing complex (RISC). An additional example ofmodulation of RNA target function is by an occupancy-based mechanismsuch as is employed naturally by microRNA. MicroRNAs are smallnon-coding RNAs that regulate the expression of protein-coding RNAs. Thebinding of an antisense compound to a microRNA prevents that microRNAfrom binding to its messenger RNA targets, and thus interferes with thefunction of the microRNA. MicroRNA mimics can enhance native microRNAfunction. Certain antisense compounds alter splicing of pre-mRNA.Regardless of the specific mechanism, sequence-specificity makesantisense compounds attractive as tools for target validation and genefunctionalization, as well as therapeutics to selectively modulate theexpression of genes involved in the pathogenesis of diseases.

Antisense technology is an effective means for modulating the expressionof one or more specific gene products and can therefore prove to beuniquely useful in a number of therapeutic, diagnostic, and researchapplications. Chemically modified nucleosides may be incorporated intoantisense compounds to enhance one or more properties, such as nucleaseresistance, pharmacokinetics or affinity for a target nucleic acid. In1998, the antisense compound, Vitravene® (fomivirsen; developed by IsisPharmaceuticals Inc., Carlsbad, Calif.) was the first antisense drug toachieve marketing clearance from the U.S. Food and Drug Administration(FDA), and is currently a treatment of cytomegalovirus (CMV)-inducedretinitis in AIDS patients.

New chemical modifications have improved the potency and efficacy ofantisense compounds, uncovering the potential for oral delivery as wellas enhancing subcutaneous administration, decreasing potential for sideeffects, and leading to improvements in patient convenience. Chemicalmodifications increasing potency of antisense compounds allowadministration of lower doses, which reduces the potential for toxicity,as well as decreasing overall cost of therapy. Modifications increasingthe resistance to degradation result in slower clearance from the body,allowing for less frequent dosing. Different types of chemicalmodifications can be combined in one compound to further optimize thecompound's efficacy.

Apolipoprotein C-III (also called APOC3, APOC-III, ApoCIII, and APOC-III) is a constituent of HDL and of triglyceride (TG)-richlipoproteins. Elevated ApoCIII levels are associated with elevated TGlevels and diseases such as cardiovascular disease, metabolic syndrome,obesity and diabetes (Chan et al., Int J Clin Pract, 2008, 62:799-809;Onat et at., Atherosclerosis, 2003, 168:81-89; Mendivil et al.,Circulation, 2011, 124:2065-2072; Mauger et al., J. Lipid Res, 2006. 47:1212-1218; Chan et al., Clin. Chem, 2002. 278-283; Ooi et al., Clin.Sci, 2008. 114: 611-624; Davidsson et al., J. Lipid Res. 2005. 46:1999-2006; Sacks et al., Circulation, 2000. 102: 1886-1892; Lee et al.,Arterioscler Thromb Vasc Biol, 2003. 23: 853-858). ApoCIII slowsclearance of TG-rich lipoproteins by inhibiting lipolysis throughinhibition of lipoprotein lipase (LPL) and through interfering withlipoprotein binding to cell-surface glycosaminoglycan matrix (Shachter,Curr. Opin. Lipidol, 2001, 12, 297-304).

Antisense technology is emerging as an effective means for reducing theexpression of certain gene products and may prove to be uniquely usefulin a number of therapeutic, diagnostic, and research applications forthe modulation of ApoCIII. Antisense compounds targeting ApoCIII andassociated methods for inhibiting ApoCIII have been previously disclosed(see e.g., U.S. Pat. No. 7,598,227, U.S. Pat. No. 7,750,141, PCTpublication WO 2004/093783, PCT publication WO 2012/149495 andPCT/US14/016546, all incorporated-by-reference herein). An antisensecompound targeting ApoCIII, ISIS-APOCIII_(Rx), has been tested in aPhase I and II clinical trials. However, no antisense compoundstargeting ApoCIII have been approved for commercial use, accordingly,there is still a need to provide patients with additional and morepotent treatment options.

SUMMARY OF THE INVENTION

In certain embodiments, the present disclosure provides conjugatedantisense compounds. In certain embodiments, the present disclosureprovides conjugated antisense compounds comprising an antisenseoligonucleotide complementary to a nucleic acid transcript. In certainembodiments, the present disclosure provides methods comprisingcontacting a cell with a conjugated antisense compound comprising anantisense oligonucleotide complementary to a nucleic acid transcript. Incertain embodiments, the present disclosure provides methods comprisingcontacting a cell with a conjugated antisense compound comprising anantisense oligonucleotide and reducing the amount or activity of anucleic acid transcript in a cell.

The asialoglycoprotein receptor (ASGP-R) has been described previously.See e.g., Park et al., PNAS vol. 102, No. 47, pp 17125-17129 (2005).Such receptors are expressed on liver cells, particularly hepatocytes.Further, it has been shown that compounds comprising clusters of threeN-acetylgalactosamine (GalNAc) ligands are capable of binding to theASGP-R, resulting in uptake of the compound into the cell. See e.g.,Khorev et al., Bioorganic and Medicinal Chemistry, 16, 9, pp 5216-5231(May 2008). Accordingly, conjugates comprising such GalNAc clusters havebeen used to facilitate uptake of certain compounds into liver cells,specifically hepatocytes. For example it has been shown that certainGalNAc-containing conjugates increase activity of duplex siRNA compoundsin liver cells in vivo. In such instances, the GalNAc-containingconjugate is typically attached to the sense strand of the siRNA duplex.Since the sense strand is discarded before the antisense strandultimately hybridizes with the target nucleic acid, there is littleconcern that the conjugate will interfere with activity. Typically, theconjugate is attached to the 3′ end of the sense strand of the siRNA.See e.g., U.S. Pat. No. 8,106,022. Certain conjugate groups describedherein are more active and/or easier to synthesize than conjugate groupspreviously described.

In certain embodiments of the present invention, conjugates are attachedto single-stranded antisense compounds, including, but not limited toRNase H based antisense compounds and antisense compounds that altersplicing of a pre-mRNA target nucleic acid. In such embodiments, theconjugate should remain attached to the antisense compound long enoughto provide benefit (improved uptake into cells) but then should eitherbe cleaved, or otherwise not interfere with the subsequent stepsnecessary for activity, such as hybridization to a target nucleic acidand interaction with RNase H or enzymes associated with splicing orsplice modulation. This balance of properties is more important in thesetting of single-stranded antisense compounds than in siRNA compounds,where the conjugate may simply be attached to the sense strand.Disclosed herein are conjugated single-stranded antisense compoundshaving improved potency in liver cells in vivo compared with the sameantisense compound lacking the conjugate. Given the required balance ofproperties for these compounds such improved potency is surprising.

In certain embodiments, conjugate groups herein comprise a cleavablemoiety. As noted, without wishing to be bound by mechanism, it islogical that the conjugate should remain on the compound long enough toprovide enhancement in uptake, but after that, it is desirable for someportion or, ideally, all of the conjugate to be cleaved, releasing theparent compound (e.g., antisense compound) in its most active form. Incertain embodiments, the cleavable moiety is a cleavable nucleoside.Such embodiments take advantage of endogenous nucleases in the cell byattaching the rest of the conjugate (the cluster) to the antisenseoligonucleotide through a nucleoside via one or more cleavable bonds,such as those of a phosphodiester linkage. In certain embodiments, thecluster is bound to the cleavable nucleoside through a phosphodiesterlinkage. In certain embodiments, the cleavable nucleoside is attached tothe antisense oligonucleotide (antisense compound) by a phosphodiesterlinkage. In certain embodiments, the conjugate group may comprise two orthree cleavable nucleosides. In such embodiments, such cleavablenucleosides are linked to one another, to the antisense compound and/orto the cluster via cleavable bonds (such as those of a phosphodiesterlinkage). Certain conjugates herein do not comprise a cleavablenucleoside and instead comprise a cleavable bond. It is shown that thatsufficient cleavage of the conjugate from the oligonucleotide isprovided by at least one bond that is vulnerable to cleavage in the cell(a cleavable bond).

In certain embodiments, conjugated antisense compounds are prodrugs.Such prodrugs are administered to an animal and are ultimatelymetabolized to a more active form. For example, conjugated antisensecompounds are cleaved to remove all or part of the conjugate resultingin the active (or more active) form of the antisense compound lackingall or some of the conjugate.

In certain embodiments, conjugates are attached at the 5′ end of anoligonucleotide. Certain such 5′-conjugates are cleaved more efficientlythan counterparts having a similar conjugate group attached at the 3′end. In certain embodiments, improved activity may correlate withimproved cleavage. In certain embodiments, oligonucleotides comprising aconjugate at the 5′ end have greater efficacy than oligonucleotidescomprising a conjugate at the 3′ end (see, for example, Examples 56, 81,83, and 84). Further, 5′-attachment allows simpler oligonucleotidesynthesis. Typically, oligonucleotides are synthesized on a solidsupport in the 3′ to 5′ direction. To make a 3′-conjugatedoligonucleotide, typically one attaches a pre-conjugated 3′ nucleosideto the solid support and then builds the oligonucleotide as usual.However, attaching that conjugated nucleoside to the solid support addscomplication to the synthesis. Further, using that approach, theconjugate is then present throughout the synthesis of theoligonucleotide and can become degraded during subsequent steps or maylimit the sorts of reactions and reagents that can be used. Using thestructures and techniques described herein for 5′-conjugatedoligonucleotides, one can synthesize the oligonucleotide using standardautomated techniques and introduce the conjugate with the final(5′-most) nucleoside or after the oligonucleotide has been cleaved fromthe solid support.

In view of the art and the present disclosure, one of ordinary skill caneasily make any of the conjugates and conjugated oligonucleotidesherein. Moreover, synthesis of certain such conjugates and conjugatedoligonucleotides disclosed herein is easier and/or requires few steps,and is therefore less expensive than that of conjugates previouslydisclosed, providing advantages in manufacturing. For example, thesynthesis of certain conjugate groups consists of fewer synthetic steps,resulting in increased yield, relative to conjugate groups previouslydescribed. Conjugate groups such as GalNAc3-10 in Example 46 andGalNAc3-7 in Example 48 are much simpler than previously describedconjugates such as those described in U.S. Pat. No. 8,106,022 or U.S.Pat. No. 7,262,177 that require assembly of more chemical intermediates.Accordingly, these and other conjugates described herein have advantagesover previously described compounds for use with any oligonucleotide,including single-stranded oligonucleotides and either strand ofdouble-stranded oligonucleotides (e.g., siRNA).

Similarly, disclosed herein are conjugate groups having only one or twoGalNAc ligands. As shown, such conjugates groups improve activity ofantisense compounds. Such compounds are much easier to prepare thanconjugates comprising three GalNAc ligands. Conjugate groups comprisingone or two GalNAc ligands may be attached to any antisense compounds,including single-stranded oligonucleotides and either strand ofdouble-stranded oligonucleotides (e.g., siRNA).

In certain embodiments, the conjugates herein do not substantially altercertain measures of tolerability. For example, it is shown herein thatconjugated antisense compounds are not more immunogenic thanunconjugated parent compounds. Since potency is improved, embodiments inwhich tolerability remains the same (or indeed even if tolerabilityworsens only slightly compared to the gains in potency) have improvedproperties for therapy.

In certain embodiments, conjugation allows one to alter antisensecompounds in ways that have less attractive consequences in the absenceof conjugation. For example, in certain embodiments, replacing one ormore phosphorothioate linkages of a fully phosphorothioate antisensecompound with phosphodiester linkages results in improvement in somemeasures of tolerability. For example, in certain instances, suchantisense compounds having one or more phosphodiester are lessimmunogenic than the same compound in which each linkage is aphosphorothioate. However, in certain instances, as shown in Example 26,that same replacement of one or more phosphorothioate linkages withphosphodiester linkages also results in reduced cellular uptake and/orloss in potency. In certain embodiments, conjugated antisense compoundsdescribed herein tolerate such change in linkages with little or no lossin uptake and potency when compared to the conjugatedfull-phosphorothioate counterpart. In fact, in certain embodiments, forexample, in Examples 44, 57, 59, and 86, oligonucleotides comprising aconjugate and at least one phosphodiester internucleoside linkageactually exhibit increased potency in vivo even relative to a fullphosphorothioate counterpart also comprising the same conjugate.Moreover, since conjugation results in substantial increases inuptake/potency a small loss in that substantial gain may be acceptableto achieve improved tolerability. Accordingly, in certain embodiments,conjugated antisense compounds comprise at least one phosphodiesterlinkage.

In certain embodiments, conjugation of antisense compounds hereinresults in increased delivery, uptake and activity in hepatocytes. Thus,more compound is delivered to liver tissue. However, in certainembodiments, that increased delivery alone does not explain the entireincrease in activity. In certain such embodiments, more compound entershepatocytes. In certain embodiments, even that increased hepatocyteuptake does not explain the entire increase in activity. In suchembodiments, productive uptake of the conjugated compound is increased.For example, as shown in Example 102, certain embodiments ofGalNAc-containing conjugates increase enrichment of antisenseoligonucleotides in hepatocytes versus non-parenchymal cells. Thisenrichment is beneficial for oligonucleotides that target genes that areexpressed in hepatocytes.

In certain embodiments, conjugated antisense compounds herein result inreduced kidney exposure. For example, as shown in Example 20, theconcentrations of antisense oligonucleotides comprising certainembodiments of GalNAc-containing conjugates are lower in the kidney thanthat of antisense oligonucleotides lacking a GalNAc-containingconjugate. This has several beneficial therapeutic implications. Fortherapeutic indications where activity in the kidney is not sought,exposure to kidney risks kidney toxicity without corresponding benefit.Moreover, high concentration in kidney typically results in loss ofcompound to the urine resulting in faster clearance. Accordingly fornon-kidney targets, kidney accumulation is undesired.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the formula:

A-B-C-DE-F)_(q)

whereinA is the antisense oligonucleotide;B is the cleavable moietyC is the conjugate linkerD is the branching groupeach E is a tether;each F is a ligand; andq is an integer between 1 and 5.

In the above diagram and in similar diagrams herein, the branching group“D” branches as many times as is necessary to accommodate the number of(E-F) groups as indicated by “q”. Thus, where q=1, the formula is:

A-B-C-D-E-F

where q=2, the formula is:

where q=3, the formula is:

where q=4, the formula is:

where q=5, the formula is:

In certain embodiments, conjugated antisense compounds are providedhaving the structure:

In certain embodiments, conjugated antisense compounds are providedhaving the structure:

In certain embodiments, conjugated antisense compounds are providedhaving the structure:

In certain embodiments, conjugated antisense compounds are providedhaving the structure:

The present disclosure provides the following non-limiting embodiments:

In embodiments having more than one of a particular variable (e.g., morethan one “m” or “n”), unless otherwise indicated, each such particularvariable is selected independently. Thus, for a structure having morethan one n, each n is selected independently, so they may or may not bethe same as one another.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting ApoCIII and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides. Incertain embodiments, the modified oligonucleotide with the conjugategroup consists of 20 linked nucleosides.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting ApoCIII and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andcomprises a nucleobase sequence complementary to an equal length portionof nucleobases 3533 to 3552 of SEQ ID NO: 3, wherein the nucleobasesequence of the modified oligonucleotide is at least 80% complementaryto SEQ ID NO: 3.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting ApoCIII and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andcomprises a nucleobase sequence complementary to an equal length portionof nucleobases 3514 to 3558 of SEQ ID NO: 3, wherein the nucleobasesequence of the modified oligonucleotide is at least 80% complementaryto SEQ ID NO: 3.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting ApoCIII and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides and hasa nucleobase sequence of any of the nucleobase sequences of SEQ ID NOs:19-96, 209-221. In certain embodiments, the conjugated modifiedoligonucleotide has a nucleobase sequence comprising at least 8contiguous nucleobases of any one of the nucleobase sequences of SEQ IDNOs: 19-96, 209-221. In certain embodiments, the compound consists ofany one of SEQ ID NOs: 19-96, 209-221 and a conjugate group.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting ApoCIII and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides and hasa nucleobase sequence of SEQ ID NO: 87. In certain embodiments, themodified oligonucleotide with the conjugate group has a nucleobasesequence comprising at least 8 contiguous nucleobases of the nucleobasesequence of SEQ ID NO: 87. In certain embodiments, the compound consistsof SEQ ID NO: 87 and a conjugate group.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises the modifiedoligonucleotide ISIS 304801 with a 5′-X, wherein X is a conjugate groupcomprising GalNAc. In certain embodiments, the antisense compoundconsists of the modified oligonucleotide ISIS 304801 with a 5′-X,wherein X is a conjugate group comprising GalNAc.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises the conjugated modifiedoligonucleotide ISIS 678354. In certain embodiments, the antisensecompound consists of the conjugated modified oligonucleotide ISIS678354.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises the conjugated modifiedoligonucleotide ISIS 678357. In certain embodiments, the antisensecompound consists of the conjugated modified oligonucleotide ISIS678357.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises a modified oligonucleotidewith the nucleobase sequence of SEQ ID NO: 87 with a 5′-GalNAc withvariability in the sugar mods of the wings. In certain embodiments, theantisense compound consists of a modified oligonucleotide with thenucleobase sequence of SEQ ID NO: 87 with a 5′-GalNAc with variabilityin the sugar mods of the wings.

Wherein either R¹ is —OCH₂CH₂OCH₃ (MOE) and R² is H; or R¹ and R²together form a bridge, wherein R¹ is —O— and R² is —CH₂—, —CH(CH₃)—, or—CH₂CH₂—, and R¹ and R² are directly connected such that the resultingbridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and —O—CH₂CH₂—;

And for each pair of R³ and R⁴ on the same ring, independently for eachring: either R³ is selected from H and —OCH₂CH₂OCH₃ and R⁴ is H; or R³and R⁴ together form a bridge, wherein R³ is —O—, and R⁴ is —CH₂—,—CH(CH₃)—, or —CH₂CH₂— and R³ and R⁴ are directly connected such thatthe resulting bridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and—O—CH₂CH₂—;

And R⁵ is selected from H and —CH₃;

And Z is selected from S⁻ and O⁻.

The present disclosure provides the following non-limiting numberedembodiments:

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the disclosure. Herein, the use of thesingular includes the plural unless specifically stated otherwise. Asused herein, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including” as well as other forms,such as “includes” and “included”, is not limiting. Also, terms such as“element” or “component” encompass both elements and componentscomprising one unit and elements and components that comprise more thanone subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

A. Definitions

Unless specific definitions are provided, the nomenclature used inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis. Certain such techniques and procedures may be foundfor example in “Carbohydrate Modifications in Antisense Research” Editedby Sangvi and Cook, American Chemical Society, Washington D.C., 1994;“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,21^(st) edition, 2005; and “Antisense Drug Technology, Principles,Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press,Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratoryManual,” 2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989,which are hereby incorporated by reference for any purpose. Wherepermitted, all patents, applications, published applications and otherpublications and other data referred to throughout in the disclosure areincorporated by reference herein in their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

As used herein, “nucleoside” means a compound comprising a nucleobasemoiety and a sugar moiety. Nucleosides include, but are not limited to,naturally occurring nucleosides (as found in DNA and RNA) and modifiednucleosides. Nucleosides may be linked to a phosphate moiety.

As used herein, “chemical modification” means a chemical difference in acompound when compared to a naturally occurring counterpart. Chemicalmodifications of oligonucleotides include nucleoside modifications(including sugar moiety modifications and nucleobase modifications) andinternucleoside linkage modifications. In reference to anoligonucleotide, chemical modification does not include differences onlyin nucleobase sequence.

As used herein, “furanosyl” means a structure comprising a 5-memberedring comprising four carbon atoms and one oxygen atom.

As used herein, “naturally occurring sugar moiety” means a ribofuranosylas found in naturally occurring RNA or a deoxyribofuranosyl as found innaturally occurring DNA.

As used herein, “sugar moiety” means a naturally occurring sugar moietyor a modified sugar moiety of a nucleoside.

As used herein, “modified sugar moiety” means a substituted sugar moietyor a sugar surrogate.

As used herein, “substituted sugar moiety” means a furanosyl that is nota naturally occurring sugar moiety. Substituted sugar moieties include,but are not limited to furanosyls comprising substituents at the2′-position, the 3′-position, the 5′-position and/or the 4′-position.Certain substituted sugar moieties are bicyclic sugar moieties.

As used herein, “2′-substituted sugar moiety” means a furanosylcomprising a substituent at the 2′-position other than H or OH. Unlessotherwise indicated, a 2′-substituted sugar moiety is not a bicyclicsugar moiety (i.e., the 2′-substituent of a 2′-substituted sugar moietydoes not form a bridge to another atom of the furanosyl ring.

As used herein, “MOE” means —OCH₂CH₂OCH₃.

As used herein, “2′-F nucleoside” refers to a nucleoside comprising asugar comprising fluorine at the 2′ position. Unless otherwiseindicated, the fluorine in a 2′-F nucleoside is in the ribo position(replacing the OH of a natural ribose).

As used herein the term “sugar surrogate” means a structure that doesnot comprise a furanosyl and that is capable of replacing the naturallyoccurring sugar moiety of a nucleoside, such that the resultingnucleoside sub-units are capable of linking together and/or linking toother nucleosides to form an oligomeric compound which is capable ofhybridizing to a complementary oligomeric compound. Such structuresinclude rings comprising a different number of atoms than furanosyl(e.g., 4, 6, or 7-membered rings); replacement of the oxygen of afuranosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); orboth a change in the number of atoms and a replacement of the oxygen.Such structures may also comprise substitutions corresponding to thosedescribed for substituted sugar moieties (e.g., 6-membered carbocyclicbicyclic sugar surrogates optionally comprising additionalsubstituents). Sugar surrogates also include more complex sugarreplacements (e.g., the non-ring systems of peptide nucleic acid). Sugarsurrogates include without limitation morpholinos, cyclohexenyls andcyclohexitols.

As used herein, “bicyclic sugar moiety” means a modified sugar moietycomprising a 4 to 7 membered ring (including but not limited to afuranosyl) comprising a bridge connecting two atoms of the 4 to 7membered ring to form a second ring, resulting in a bicyclic structure.In certain embodiments, the 4 to 7 membered ring is a sugar ring. Incertain embodiments the 4 to 7 membered ring is a furanosyl. In certainsuch embodiments, the bridge connects the 2′-carbon and the 4′-carbon ofthe furanosyl.

As used herein, “nucleotide” means a nucleoside further comprising aphosphate linking group. As used herein, “linked nucleosides” may or maynot be linked by phosphate linkages and thus includes, but is notlimited to “linked nucleotides.” As used herein, “linked nucleosides”are nucleosides that are connected in a continuous sequence (i.e. noadditional nucleosides are present between those that are linked).

As used herein, “nucleic acid” refers to molecules composed of monomericnucleotides. A nucleic acid includes ribonucleic acids (RNA),deoxyribonucleic acids (DNA), single-stranded nucleic acids (ssDNA),double-stranded nucleic acids (dsDNA), small interfering ribonucleicacids (siRNA), and microRNAs (miRNA). A nucleic acid may also compriseany combination of these elements in a single molecule.

As used herein, “nucleotide” means a nucleoside further comprising aphosphate linking group. As used herein, “linked nucleosides” may or maynot be linked by phosphate linkages and thus includes, but is notlimited to “linked nucleotides.” As used herein, “linked nucleosides”are nucleosides that are connected in a continuous sequence (i.e. noadditional nucleosides are present between those that are linked).

As used herein, “nucleobase” means a group of atoms that can be linkedto a sugar moiety to create a nucleoside that is capable ofincorporation into an oligonucleotide, and wherein the group of atoms iscapable of bonding with a complementary naturally occurring nucleobaseof another oligonucleotide or nucleic acid. Nucleobases may be naturallyoccurring or may be modified. As used herein, “nucleobase sequence”means the order of contiguous nucleobases independent of any sugar,linkage, or nucleobase modification.

As used herein the terms, “unmodified nucleobase” or “naturallyoccurring nucleobase” means the naturally occurring heterocyclicnucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G),and the pyrimidine bases thymine (T), cytosine (C) (including 5-methylC), and uracil (U).

As used herein, “modified nucleobase” means any nucleobase that is not anaturally occurring nucleobase.

As used herein, “modified nucleoside” means a nucleoside comprising atleast one chemical modification compared to naturally occurring RNA orDNA nucleosides. Modified nucleosides comprise a modified sugar moietyand/or a modified nucleobase.

As used herein, “bicyclic nucleoside” or “BNA” means a nucleosidecomprising a bicyclic sugar moiety.

As used herein, “constrained ethyl nucleoside” or “cEt” means anucleoside comprising a bicyclic sugar moiety comprising a4′-CH(CH₃)—O-2′bridge.

As used herein, “locked nucleic acid nucleoside” or “LNA” means anucleoside comprising a bicyclic sugar moiety comprising a4′-CH₂—O-2′bridge.

As used herein, “2′-substituted nucleoside” means a nucleosidecomprising a substituent at the 2′-position other than H or OH. Unlessotherwise indicated, a 2′-substituted nucleoside is not a bicyclicnucleoside.

As used herein, “deoxynucleoside” means a nucleoside comprising 2′-Hfuranosyl sugar moiety, as found in naturally occurringdeoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleosidemay comprise a modified nucleobase or may comprise an RNA nucleobase(e.g., uracil).

As used herein, “oligonucleotide” means a compound comprising aplurality of linked nucleosides. In certain embodiments, anoligonucleotide comprises one or more unmodified ribonucleosides (RNA)and/or unmodified deoxyribonucleosides (DNA) and/or one or more modifiednucleosides.

As used herein “oligonucleoside” means an oligonucleotide in which noneof the internucleoside linkages contains a phosphorus atom. As usedherein, oligonucleotides include oligonucleosides.

As used herein, “modified oligonucleotide” means an oligonucleotidecomprising at least one modified nucleoside and/or at least one modifiedinternucleoside linkage.

As used herein, “linkage” or “linking group” means a group of atoms thatlink together two or more other groups of atoms.

As used herein “internucleoside linkage” means a covalent linkagebetween adjacent nucleosides in an oligonucleotide.

As used herein “naturally occurring internucleoside linkage” means a 3′to 5′ phosphodiester linkage. As used herein, “modified internucleosidelinkage” means any internucleoside linkage other than a naturallyoccurring internucleoside linkage.

As used herein, “terminal internucleoside linkage” means the linkagebetween the last two nucleosides of an oligonucleotide or defined regionthereof.

As used herein, “phosphorus linking group” means a linking groupcomprising a phosphorus atom. Phosphorus linking groups include withoutlimitation groups having the formula:

wherein:

R_(a) and R_(d) are each, independently, O, S, CH₂, NH, or NJ₁ whereinJ₁ is C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

R_(b) is O or S;

R_(c) is OH, SH, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy,substituted C₁-C₆ alkoxy, amino or substituted amino; and

J₁ is R_(b) is O or S.

Phosphorus linking groups include without limitation, phosphodiester,phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate,phosphorothioamidate, thionoalkylphosphonate, phosphotriesters,thionoalkylphosphotriester and boranophosphate.

As used herein, “internucleoside phosphorus linking group” means aphosphorus linking group that directly links two nucleosides.

As used herein, “non-internucleoside phosphorus linking group” means aphosphorus linking group that does not directly link two nucleosides. Incertain embodiments, a non-internucleoside phosphorus linking grouplinks a nucleoside to a group other than a nucleoside. In certainembodiments, a non-internucleoside phosphorus linking group links twogroups, neither of which is a nucleoside.

As used herein, “neutral linking group” means a linking group that isnot charged. Neutral linking groups include without limitationphosphotriesters, methylphosphonates, MMI (—CH₂—N(CH₃)—O—), amide-3(—CH₂—C(═O)—N(H)—), amide-4 (—CH₂—N(H)—C(═O)—), formacetal (—O—CH₂—O—),and thioformacetal (—S—CH₂—O—). Further neutral linking groups includenonionic linkages comprising siloxane (dialkylsiloxane), carboxylateester, carboxamide, sulfide, sulfonate ester and amides (See forexample: Carbohydrate Modifications in Antisense Research; Y. S. Sanghviand P. D. Cook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp.40-65)). Further neutral linking groups include nonionic linkagescomprising mixed N, O, S and CH₂ component parts.

As used herein, “internucleoside neutral linking group” means a neutrallinking group that directly links two nucleosides.

As used herein, “non-internucleoside neutral linking group” means aneutral linking group that does not directly link two nucleosides. Incertain embodiments, a non-internucleoside neutral linking group links anucleoside to a group other than a nucleoside. In certain embodiments, anon-internucleoside neutral linking group links two groups, neither ofwhich is a nucleoside.

As used herein, “oligomeric compound” means a polymeric structurecomprising two or more sub-structures. In certain embodiments, anoligomeric compound comprises an oligonucleotide. In certainembodiments, an oligomeric compound comprises one or more conjugategroups and/or terminal groups. In certain embodiments, an oligomericcompound consists of an oligonucleotide. Oligomeric compounds alsoinclude naturally occurring nucleic acids. In certain embodiments, anoligomeric compound comprises a backbone of one or more linked monomericsubunits where each linked monomeric subunit is directly or indirectlyattached to a heterocyclic base moiety. In certain embodiments,oligomeric compounds may also include monomeric subunits that are notlinked to a heterocyclic base moiety, thereby providing abasic sites.

In certain embodiments, the linkages joining the monomeric subunits, thesugar moieties or surrogates and the heterocyclic base moieties can beindependently modified. In certain embodiments, the linkage-sugar unit,which may or may not include a heterocyclic base, may be substitutedwith a mimetic such as the monomers in peptide nucleic acids.

As used herein, “terminal group” means one or more atom attached toeither, or both, the 3′ end or the 5′ end of an oligonucleotide. Incertain embodiments a terminal group is a conjugate group. In certainembodiments, a terminal group comprises one or more terminal groupnucleosides.

As used herein, “conjugate” or “conjugate group” means an atom or groupof atoms bound to an oligonucleotide or oligomeric compound. In general,conjugate groups modify one or more properties of the compound to whichthey are attached, including, but not limited to pharmacodynamic,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and/or clearance properties.

As used herein, “conjugate linker” or “linker” in the context of aconjugate group means a portion of a conjugate group comprising any atomor group of atoms and which covalently link (1) an oligonucleotide toanother portion of the conjugate group or (2) two or more portions ofthe conjugate group.

Conjugate groups are shown herein as radicals, providing a bond forforming covalent attachment to an oligomeric compound such as anantisense oligonucleotide. In certain embodiments, the point ofattachment on the oligomeric compound is the 3′-oxygen atom of the3′-hydroxyl group of the 3′ terminal nucleoside of the oligomericcompound. In certain embodiments the point of attachment on theoligomeric compound is the 5′-oxygen atom of the 5′-hydroxyl group ofthe 5′ terminal nucleoside of the oligomeric compound. In certainembodiments, the bond for forming attachment to the oligomeric compoundis a cleavable bond. In certain such embodiments, such cleavable bondconstitutes all or part of a cleavable moiety.

In certain embodiments, conjugate groups comprise a cleavable moiety(e.g., a cleavable bond or cleavable nucleoside) and a carbohydratecluster portion, such as a GalNAc cluster portion. Such carbohydratecluster portion comprises: a targeting moiety and, optionally, aconjugate linker. In certain embodiments, the carbohydrate clusterportion is identified by the number and identity of the ligand. Forexample, in certain embodiments, the carbohydrate cluster portioncomprises 3 GalNAc groups and is designated “GalNAc3”. In certainembodiments, the carbohydrate cluster portion comprises 4 GalNAc groupsand is designated “GalNAc4”. Specific carbohydrate cluster portions(having specific tether, branching and conjugate linker groups) aredescribed herein and designated by Roman numeral followed by subscript“a”. Accordingly “GalNac3-1_(a)” refers to a specific carbohydratecluster portion of a conjugate group having 3 GalNac groups andspecifically identified tether, branching and linking groups. Suchcarbohydrate cluster fragment is attached to an oligomeric compound viaa cleavable moiety, such as a cleavable bond or cleavable nucleoside.

As used herein, “cleavable moiety” means a bond or group that is capableof being split under physiological conditions. In certain embodiments, acleavable moiety is cleaved inside a cell or sub-cellular compartments,such as a lysosome. In certain embodiments, a cleavable moiety iscleaved by endogenous enzymes, such as nucleases. In certainembodiments, a cleavable moiety comprises a group of atoms having one,two, three, four, or more than four cleavable bonds.

As used herein, “cleavable bond” means any chemical bond capable ofbeing split. In certain embodiments, a cleavable bond is selected fromamong: an amide, a polyamide, an ester, an ether, one or both esters ofa phosphodiester, a phosphate ester, a carbamate, a di-sulfide, or apeptide.

As used herein, “carbohydrate cluster” means a compound having one ormore carbohydrate residues attached to a scaffold or linker group. (see,e.g., Maier et al., “Synthesis of Antisense Oligonucleotides Conjugatedto a Multivalent Carbohydrate Cluster for Cellular Targeting,”Bioconjugate Chemistry, 2003, (14): 18-29, which is incorporated hereinby reference in its entirety, or Rensen et al., “Design and Synthesis ofNovel N-Acetylgalactosamine-Terminated Glycolipids for Targeting ofLipoproteins to the Hepatic Asiaglycoprotein Receptor,” J. Med. Chem.2004, (47): 5798-5808, for examples of carbohydrate conjugate clusters).

As used herein, “modified carbohydrate” means any carbohydrate havingone or more chemical modifications relative to naturally occurringcarbohydrates.

As used herein, “carbohydrate derivative” means any compound which maybe synthesized using a carbohydrate as a starting material orintermediate.

As used herein, “carbohydrate” means a naturally occurring carbohydrate,a modified carbohydrate, or a carbohydrate derivative.

As used herein “protecting group” means any compound or protecting groupknown to those having skill in the art. Non-limiting examples ofprotecting groups may be found in “Protective Groups in OrganicChemistry”, T. W. Greene, P. G. M. Wuts, ISBN O-471-62301-6, John Wiley& Sons, Inc, New York, which is incorporated herein by reference in itsentirety.

As used herein, “single-stranded” means an oligomeric compound that isnot hybridized to its complement and which lacks sufficientself-complementarity to form a stable self-duplex.

As used herein, “double stranded” means a pair of oligomeric compoundsthat are hybridized to one another or a single self-complementaryoligomeric compound that forms a hairpin structure. In certainembodiments, a double-stranded oligomeric compound comprises a first anda second oligomeric compound.

As used herein, “antisense compound” means a compound comprising orconsisting of an oligonucleotide at least a portion of which iscomplementary to a target nucleic acid to which it is capable ofhybridizing, resulting in at least one antisense activity.

As used herein, “antisense activity” means any detectable and/ormeasurable change attributable to the hybridization of an antisensecompound to its target nucleic acid. In certain embodiments, antisenseactivity includes modulation of the amount or activity of a targetnucleic acid transcript (e.g. mRNA). In certain embodiments, antisenseactivity includes modulation of the splicing of pre-mRNA.

As used herein, “RNase H based antisense compound” means an antisensecompound wherein at least some of the antisense activity of theantisense compound is attributable to hybridization of the antisensecompound to a target nucleic acid and subsequent cleavage of the targetnucleic acid by RNase H.

As used herein, “RISC based antisense compound” means an antisensecompound wherein at least some of the antisense activity of theantisense compound is attributable to the RNA Induced Silencing Complex(RISC).

As used herein, “detecting” or “measuring” means that a test or assayfor detecting or measuring is performed. Such detection and/or measuringmay result in a value of zero. Thus, if a test for detection ormeasuring results in a finding of no activity (activity of zero), thestep of detecting or measuring the activity has nevertheless beenperformed.

As used herein, “detectable and/or measurable activity” means astatistically significant activity that is not zero.

As used herein, “essentially unchanged” means little or no change in aparticular parameter, particularly relative to another parameter whichchanges much more. In certain embodiments, a parameter is essentiallyunchanged when it changes less than 5%. In certain embodiments, aparameter is essentially unchanged if it changes less than two-foldwhile another parameter changes at least ten-fold. For example, incertain embodiments, an antisense activity is a change in the amount ofa target nucleic acid. In certain such embodiments, the amount of anon-target nucleic acid is essentially unchanged if it changes much lessthan the target nucleic acid does, but the change need not be zero.

As used herein, “expression” means the process by which a geneultimately results in a protein. Expression includes, but is not limitedto, transcription, post-transcriptional modification (e.g., splicing,polyadenylation, addition of 5′-cap), and translation.

As used herein, “target nucleic acid” means a nucleic acid molecule towhich an antisense compound is intended to hybridize to result in adesired antisense activity. Antisense oligonucleotides have sufficientcomplementarity to their target nucleic acids to allow hybridizationunder physiological conditions.

As used herein, “nucleobase complementarity” or “complementarity” whenin reference to nucleobases means a nucleobase that is capable of basepairing with another nucleobase. For example, in DNA, adenine (A) iscomplementary to thymine (T). For example, in RNA, adenine (A) iscomplementary to uracil (U). In certain embodiments, complementarynucleobase means a nucleobase of an antisense compound that is capableof base pairing with a nucleobase of its target nucleic acid. Forexample, if a nucleobase at a certain position of an antisense compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, then the position of hydrogen bonding betweenthe oligonucleotide and the target nucleic acid is considered to becomplementary at that nucleobase pair. Nucleobases comprising certainmodifications may maintain the ability to pair with a counterpartnucleobase and thus, are still capable of nucleobase complementarity.

As used herein, “non-complementary” in reference to nucleobases means apair of nucleobases that do not form hydrogen bonds with one another.

As used herein, “complementary” in reference to oligomeric compounds(e.g., linked nucleosides, oligonucleotides, or nucleic acids) means thecapacity of such oligomeric compounds or regions thereof to hybridize toanother oligomeric compound or region thereof through nucleobasecomplementarity. Complementary oligomeric compounds need not havenucleobase complementarity at each nucleoside. Rather, some mismatchesare tolerated. In certain embodiments, complementary oligomericcompounds or regions are complementary at 70% of the nucleobases (70%complementary). In certain embodiments, complementary oligomericcompounds or regions are 80% complementary. In certain embodiments,complementary oligomeric compounds or regions are 90% complementary. Incertain embodiments, complementary oligomeric compounds or regions are95% complementary. In certain embodiments, complementary oligomericcompounds or regions are 100% complementary.

As used herein, “mismatch” means a nucleobase of a first oligomericcompound that is not capable of pairing with a nucleobase at acorresponding position of a second oligomeric compound, when the firstand second oligomeric compound are aligned. Either or both of the firstand second oligomeric compounds may be oligonucleotides.

As used herein, “hybridization” means the pairing of complementaryoligomeric compounds (e.g., an antisense compound and its target nucleicacid). While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleobases.

As used herein, “specifically hybridizes” means the ability of anoligomeric compound to hybridize to one nucleic acid site with greateraffinity than it hybridizes to another nucleic acid site.

As used herein, “fully complementary” in reference to an oligonucleotideor portion thereof means that each nucleobase of the oligonucleotide orportion thereof is capable of pairing with a nucleobase of acomplementary nucleic acid or contiguous portion thereof. Thus, a fullycomplementary region comprises no mismatches or unhybridized nucleobasesin either strand.

As used herein, “percent complementarity” means the percentage ofnucleobases of an oligomeric compound that are complementary to anequal-length portion of a target nucleic acid. Percent complementarityis calculated by dividing the number of nucleobases of the oligomericcompound that are complementary to nucleobases at correspondingpositions in the target nucleic acid by the total length of theoligomeric compound.

As used herein, “percent identity” means the number of nucleobases in afirst nucleic acid that are the same type (independent of chemicalmodification) as nucleobases at corresponding positions in a secondnucleic acid, divided by the total number of nucleobases in the firstnucleic acid.

As used herein, “modulation” means a change of amount or quality of amolecule, function, or activity when compared to the amount or qualityof a molecule, function, or activity prior to modulation. For example,modulation includes the change, either an increase (stimulation orinduction) or a decrease (inhibition or reduction) in gene expression.As a further example, modulation of expression can include a change insplice site selection of pre-mRNA processing, resulting in a change inthe absolute or relative amount of a particular splice-variant comparedto the amount in the absence of modulation.

As used herein, “chemical motif” means a pattern of chemicalmodifications in an oligonucleotide or a region thereof. Motifs may bedefined by modifications at certain nucleosides and/or at certainlinking groups of an oligonucleotide.

As used herein, “nucleoside motif” means a pattern of nucleosidemodifications in an oligonucleotide or a region thereof. The linkages ofsuch an oligonucleotide may be modified or unmodified. Unless otherwiseindicated, motifs herein describing only nucleosides are intended to benucleoside motifs. Thus, in such instances, the linkages are notlimited.

As used herein, “sugar motif” means a pattern of sugar modifications inan oligonucleotide or a region thereof.

As used herein, “linkage motif” means a pattern of linkage modificationsin an oligonucleotide or region thereof. The nucleosides of such anoligonucleotide may be modified or unmodified. Unless otherwiseindicated, motifs herein describing only linkages are intended to belinkage motifs. Thus, in such instances, the nucleosides are notlimited.

As used herein, “nucleobase modification motif” means a pattern ofmodifications to nucleobases along an oligonucleotide. Unless otherwiseindicated, a nucleobase modification motif is independent of thenucleobase sequence.

As used herein, “sequence motif” means a pattern of nucleobases arrangedalong an oligonucleotide or portion thereof. Unless otherwise indicated,a sequence motif is independent of chemical modifications and thus mayhave any combination of chemical modifications, including no chemicalmodifications.

As used herein, “type of modification” in reference to a nucleoside or anucleoside of a “type” means the chemical modification of a nucleosideand includes modified and unmodified nucleosides. Accordingly, unlessotherwise indicated, a “nucleoside having a modification of a firsttype” may be an unmodified nucleoside.

As used herein, “differently modified” mean chemical modifications orchemical substituents that are different from one another, includingabsence of modifications. Thus, for example, a MOE nucleoside and anunmodified DNA nucleoside are “differently modified,” even though theDNA nucleoside is unmodified. Likewise, DNA and RNA are “differentlymodified,” even though both are naturally-occurring unmodifiednucleosides. Nucleosides that are the same but for comprising differentnucleobases are not differently modified. For example, a nucleosidecomprising a 2′-OMe modified sugar and an unmodified adenine nucleobaseand a nucleoside comprising a 2′-OMe modified sugar and an unmodifiedthymine nucleobase are not differently modified.

As used herein, “the same type of modifications” refers to modificationsthat are the same as one another, including absence of modifications.Thus, for example, two unmodified DNA nucleosides have “the same type ofmodification,” even though the DNA nucleoside is unmodified. Suchnucleosides having the same type modification may comprise differentnucleobases.

As used herein, “separate regions” means portions of an oligonucleotidewherein the chemical modifications or the motif of chemicalmodifications of any neighboring portions include at least onedifference to allow the separate regions to be distinguished from oneanother.

As used herein, “pharmaceutically acceptable carrier or diluent” meansany substance suitable for use in administering to an animal. In certainembodiments, a pharmaceutically acceptable carrier or diluent is sterilesaline. In certain embodiments, such sterile saline is pharmaceuticalgrade saline.

As used herein the term “metabolic disorder” means a disease orcondition principally characterized by dysregulation of metabolism—thecomplex set of chemical reactions associated with breakdown of food toproduce energy.

As used herein, the term “cardiovascular disorder” means a disease orcondition principally characterized by impaired function of the heart orblood vessels.

As used herein the term “mono or polycyclic ring system” is meant toinclude all ring systems selected from single or polycyclic radical ringsystems wherein the rings are fused or linked and is meant to beinclusive of single and mixed ring systems individually selected fromaliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl,heterocyclic, heteroaryl, heteroaromatic and heteroarylalkyl. Such monoand poly cyclic structures can contain rings that each have the samelevel of saturation or each, independently, have varying degrees ofsaturation including fully saturated, partially saturated or fullyunsaturated. Each ring can comprise ring atoms selected from C, N, O andS to give rise to heterocyclic rings as well as rings comprising only Cring atoms which can be present in a mixed motif such as for examplebenzimidazole wherein one ring has only carbon ring atoms and the fusedring has two nitrogen atoms. The mono or polycyclic ring system can befurther substituted with substituent groups such as for examplephthalimide which has two ═O groups attached to one of the rings. Monoor polycyclic ring systems can be attached to parent molecules usingvarious strategies such as directly through a ring atom, fused throughmultiple ring atoms, through a substituent group or through abifunctional linking moiety.

As used herein, “prodrug” means an inactive or less active form of acompound which, when administered to a subject, is metabolized to formthe active, or more active, compound (e.g., drug).

As used herein, “substituent” and “substituent group,” means an atom orgroup that replaces the atom or group of a named parent compound. Forexample a substituent of a modified nucleoside is any atom or group thatdiffers from the atom or group found in a naturally occurring nucleoside(e.g., a modified 2′-substituent is any atom or group at the 2′-positionof a nucleoside other than H or OH). Substituent groups can be protectedor unprotected. In certain embodiments, compounds of the presentdisclosure have substituents at one or at more than one position of theparent compound. Substituents may also be further substituted with othersubstituent groups and may be attached directly or via a linking groupsuch as an alkyl or hydrocarbyl group to a parent compound.

Likewise, as used herein, “substituent” in reference to a chemicalfunctional group means an atom or group of atoms that differs from theatom or a group of atoms normally present in the named functional group.In certain embodiments, a substituent replaces a hydrogen atom of thefunctional group (e.g., in certain embodiments, the substituent of asubstituted methyl group is an atom or group other than hydrogen whichreplaces one of the hydrogen atoms of an unsubstituted methyl group).Unless otherwise indicated, groups amenable for use as substituentsinclude without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl,acyl (—C(O)R_(aa)), carboxyl (—C(O)O—R_(aa)), aliphatic groups,alicyclic groups, alkoxy, substituted oxy (—O—R_(aa)), aryl, aralkyl,heterocyclic radical, heteroaryl, heteroarylalkyl, amino(—N(R_(bb))(R_(cc))), imino(=NR_(bb)), amido (—C(O)N—(R_(bb))(R_(cc)) or—N(R_(bb))C(O)R_(aa)), azido (—N₃), nitro (—NO₂), cyano (—CN), carbamido(—OC(O)N(R_(bb))(R_(cc)) or —N(R_(bb))C(O)OR_(aa)), ureido(—N(R_(bb))C(O)N(R_(bb))(R_(cc))), thioureido(—N(R_(bb))C(S)N(R_(bb))(R_(cc))), guanidinyl(—N(R_(bb))C(═NR_(bb))N(R_(bb))(R_(cc))), amidinyl(—C(═NR_(bb))N(R_(bb))(R_(cc)) or —N(R_(bb))C(═NR_(bb))(R_(aa))), thiol(—SR_(bb)), sulfinyl (—S(O)R_(bb)), sulfonyl (—S(O)₂R_(bb)) andsulfonamidyl (—S(O)₂N(R_(bb))(R_(cc)) or —N(R_(bb))S(O)₂R_(bb)). Whereineach R_(aa), R_(bb) and R_(cc) is, independently, H, an optionallylinked chemical functional group or a further substituent group with apreferred list including without limitation, alkyl, alkenyl, alkynyl,aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic,heterocyclic and heteroarylalkyl. Selected substituents within thecompounds described herein are present to a recursive degree.

As used herein, “alkyl,” as used herein, means a saturated straight orbranched hydrocarbon radical containing up to twenty four carbon atoms.Examples of alkyl groups include without limitation, methyl, ethyl,propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.Alkyl groups typically include from 1 to about 24 carbon atoms, moretypically from 1 to about 12 carbon atoms (C₁-C₁₂ alkyl) with from 1 toabout 6 carbon atoms being more preferred.

As used herein, “alkenyl,” means a straight or branched hydrocarbonchain radical containing up to twenty four carbon atoms and having atleast one carbon-carbon double bond. Examples of alkenyl groups includewithout limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl,dienes such as 1,3-butadiene and the like. Alkenyl groups typicallyinclude from 2 to about 24 carbon atoms, more typically from 2 to about12 carbon atoms with from 2 to about 6 carbon atoms being morepreferred. Alkenyl groups as used herein may optionally include one ormore further substituent groups.

As used herein, “alkynyl,” means a straight or branched hydrocarbonradical containing up to twenty four carbon atoms and having at leastone carbon-carbon triple bond. Examples of alkynyl groups include,without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like.Alkynyl groups typically include from 2 to about 24 carbon atoms, moretypically from 2 to about 12 carbon atoms with from 2 to about 6 carbonatoms being more preferred. Alkynyl groups as used herein may optionallyinclude one or more further substituent groups.

As used herein, “acyl,” means a radical formed by removal of a hydroxylgroup from an organic acid and has the general Formula —C(O)—X where Xis typically aliphatic, alicyclic or aromatic. Examples includealiphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromaticsulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphaticphosphates and the like. Acyl groups as used herein may optionallyinclude further substituent groups.

As used herein, “alicyclic” means a cyclic ring system wherein the ringis aliphatic. The ring system can comprise one or more rings wherein atleast one ring is aliphatic. Preferred alicyclics include rings havingfrom about 5 to about 9 carbon atoms in the ring. Alicyclic as usedherein may optionally include further substituent groups.

As used herein, “aliphatic” means a straight or branched hydrocarbonradical containing up to twenty four carbon atoms wherein the saturationbetween any two carbon atoms is a single, double or triple bond. Analiphatic group preferably contains from 1 to about 24 carbon atoms,more typically from 1 to about 12 carbon atoms with from 1 to about 6carbon atoms being more preferred. The straight or branched chain of analiphatic group may be interrupted with one or more heteroatoms thatinclude nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groupsinterrupted by heteroatoms include without limitation, polyalkoxys, suchas polyalkylene glycols, polyamines, and polyimines. Aliphatic groups asused herein may optionally include further substituent groups.

As used herein, “alkoxy” means a radical formed between an alkyl groupand an oxygen atom wherein the oxygen atom is used to attach the alkoxygroup to a parent molecule. Examples of alkoxy groups include withoutlimitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy,tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groupsas used herein may optionally include further substituent groups.

As used herein, “aminoalkyl” means an amino substituted C₁-C₁₂ alkylradical. The alkyl portion of the radical forms a covalent bond with aparent molecule. The amino group can be located at any position and theaminoalkyl group can be substituted with a further substituent group atthe alkyl and/or amino portions.

As used herein, “aralkyl” and “arylalkyl” mean an aromatic group that iscovalently linked to a C₁-C₁₂ alkyl radical. The alkyl radical portionof the resulting aralkyl (or arylalkyl) group forms a covalent bond witha parent molecule. Examples include without limitation, benzyl,phenethyl and the like. Aralkyl groups as used herein may optionallyinclude further substituent groups attached to the alkyl, the aryl orboth groups that form the radical group.

As used herein, “aryl” and “aromatic” mean a mono- or polycycliccarbocyclic ring system radicals having one or more aromatic rings.Examples of aryl groups include without limitation, phenyl, naphthyl,tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ringsystems have from about 5 to about 20 carbon atoms in one or more rings.Aryl groups as used herein may optionally include further substituentgroups.

As used herein, “halo” and “halogen,” mean an atom selected fromfluorine, chlorine, bromine and iodine.

As used herein, “heteroaryl,” and “heteroaromatic,” mean a radicalcomprising a mono- or poly-cyclic aromatic ring, ring system or fusedring system wherein at least one of the rings is aromatic and includesone or more heteroatoms. Heteroaryl is also meant to include fused ringsystems including systems where one or more of the fused rings containno heteroatoms. Heteroaryl groups typically include one ring atomselected from sulfur, nitrogen or oxygen. Examples of heteroaryl groupsinclude without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl,benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroarylradicals can be attached to a parent molecule directly or through alinking moiety such as an aliphatic group or hetero atom. Heteroarylgroups as used herein may optionally include further substituent groups.

As used herein, “conjugate compound” means any atoms, group of atoms, orgroup of linked atoms suitable for use as a conjugate group. In certainembodiments, conjugate compounds may possess or impart one or moreproperties, including, but not limited to pharmacodynamic,pharmacokinetic, binding, absorption, cellular distribution, cellularuptake, charge and/or clearance properties.

As used herein, unless otherwise indicated or modified, the term“double-stranded” refers to two separate oligomeric compounds that arehybridized to one another. Such double stranded compounds may have oneor more or non-hybridizing nucleosides at one or both ends of one orboth strands (overhangs) and/or one or more internal non-hybridizingnucleosides (mismatches) provided there is sufficient complementarity tomaintain hybridization under physiologically relevant conditions.

As used herein, “5′ target site” refers to the nucleotide of a targetnucleic acid which is complementary to the 5′-most nucleotide of aparticular antisense compound.

As used herein, “about” means within ±10% of a value. For example, if itis stated, “a marker may be increased by about 50%”, it is implied thatthe marker may be increased between 45%-55%.

As used herein, “administered concomitantly” refers to theco-administration of two agents in any manner in which thepharmacological effects of both are manifest in the patient at the sametime. Concomitant administration does not require that both agents beadministered in a single pharmaceutical composition, in the same dosageform, or by the same route of administration. The effects of both agentsneed not manifest themselves at the same time. The effects need only beoverlapping for a period of time and need not be coextensive.

As used herein, “administering” or “administration” means providing apharmaceutical agent to an individual, and includes, but is not limitedto, administering by a medical professional and self-administering.Administration of a pharmaceutical agent to an individual can becontinuous, chronic, short or intermittent. Administration canparenteral or non-parenteral.

As used herein, “agent” means an active substance that can provide atherapeutic benefit when administered to an animal. “First agent” meansa therapeutic compound of the invention. For example, a first agent canbe an antisense oligonucleotide targeting apoCIII. “Second agent” meansa second therapeutic compound of the invention (e.g. a second antisenseoligonucleotide targeting apoCIII) and/or a non-apoCIII therapeuticcompound.

As used herein, “amelioration” or “ameliorate” or “ameliorating” refersto a lessening of at least one indicator, sign, or symptom of anassociated disease, disorder, or condition. The severity of indicatorscan be determined by subjective or objective measures, which are knownto those skilled in the art.

As used herein, “animal” refers to a human or non-human animal,including, but not limited to, mice, rats, rabbits, dogs, cats, pigs,and non-human primates, including, but not limited to, monkeys andchimpanzees.

As used herein, “ApoCIII”, “Apolipoprotein C-III” or “ApoC3” means anynucleic acid or protein sequence encoding ApoCIII. For example, incertain embodiments, an ApoCIII includes a DNA sequence encodingApoCIII, a RNA sequence transcribed from DNA encoding ApoCIII (includinggenomic DNA comprising introns and exons), a mRNA sequence encodingApoCIII, or a peptide sequence encoding ApoCIII.

As used herein, “ApoCIII nucleic acid” means any nucleic acid encodingApoCIII. For example, in certain embodiments, an ApoCIII nucleic acidincludes a DNA sequence encoding ApoCIII, a RNA sequence transcribedfrom DNA encoding ApoCIII (including genomic DNA comprising introns andexons), and a mRNA sequence encoding ApoCIII.

As used herein, “ApoCIII specific inhibitor” refers to any agent capableof specifically inhibiting the expression of ApoCIII mRNA and/or theexpression or activity of ApoCIII protein at the molecular level. Forexample, ApoCIII specific inhibitors include nucleic acids (includingantisense compounds), peptides, antibodies, small molecules, and otheragents capable of inhibiting the expression of ApoCIII mRNA and/or

ApoCIII protein. In certain embodiments, the nucleic acid is anantisense compound. In certain embodiments, the antisense compound is aan oligonucleotide targeting ApoCIII. In certain embodiments, theoligonucleotide targeting ApoCIII is a modified oligonucleotidetargeting ApoCIII. In certain embodiments, the oligonucleotide targetingApoCIII is a modified oligonucleotide targeting ApoCIII with a conjugategroup. In certain embodiments, the oligonucleotide targeting ApoCIII hasa sequence as shown in SEQ ID NOs:19-96, 209-221 or another sequence(for example, such as those disclosed in PCT Publication WO 2004/093783or PCT Publication WO 2012/149495, all incorporated-by-referenceherein). In certain embodiments, by specifically modulating ApoCIII mRNAlevel and/or ApoCIII protein expression, ApoCIII specific inhibitors mayaffect components of the lipogenic or glucogenic pathway. Similarly, incertain embodiments, ApoCIII specific inhibitors may affect othermolecular processes in an animal.

As used herein, “ApoCIII mRNA” means a mRNA encoding an ApoCIII protein.

As used herein, “ApoCIII protein” means any protein sequence encodingApoCIII.

As used herein, “atherosclerosis” means a hardening of the arteriesaffecting large and medium-sized arteries and is characterized by thepresence of fatty deposits. The fatty deposits are called “atheromas” or“plaques,” which consist mainly of cholesterol and other fats, calciumand scar tissue, and damage the lining of arteries.

As used herein, “coronary heart disease (CHD)” means a narrowing of thesmall blood vessels that supply blood and oxygen to the heart, which isoften a result of atherosclerosis.

As used herein, “diabetes mellitus” or “diabetes” is a syndromecharacterized by disordered metabolism and abnormally high blood sugar(hyperglycemia) resulting from insufficient levels of insulin or reducedinsulin sensitivity. The characteristic symptoms are excessive urineproduction (polyuria) due to high blood glucose levels, excessive thirstand increased fluid intake (polydipsia) attempting to compensate forincreased urination, blurred vision due to high blood glucose effects onthe eye's optics, unexplained weight loss, and lethargy.

As used herein, “diabetic dyslipidemia” or “type 2 diabetes withdyslipidemia” means a condition characterized by Type 2 diabetes,reduced HDL-C, elevated triglycerides (TG), and elevated small, denseLDL particles.

As used herein, “diluent” means an ingredient in a composition thatlacks pharmacological activity, but is pharmaceutically necessary ordesirable. For example, the diluent in an injected composition can be aliquid, e.g. saline solution.

As used herein, “dyslipidemia” refers to a disorder of lipid and/orlipoprotein metabolism, including lipid and/or lipoproteinoverproduction or deficiency. Dyslipidemias can be manifested byelevation of lipids such as chylomicron, cholesterol and triglyceridesas well as lipoproteins such as low-density lipoprotein (LDL)cholesterol.

As used herein, “dosage unit” means a form in which a pharmaceuticalagent is provided, e.g. pill, tablet, or other dosage unit known in theart. In certain embodiments, a dosage unit is a vial containinglyophilized antisense oligonucleotide. In certain embodiments, a dosageunit is a vial containing reconstituted antisense oligonucleotide.

As used herein, “dose” means a specified quantity of a pharmaceuticalagent provided in a single administration, or in a specified timeperiod. In certain embodiments, a dose can be administered in one, two,or more boluses, tablets, or injections. For example, in certainembodiments where subcutaneous administration is desired, the desireddose requires a volume not easily accommodated by a single injection,therefore, two or more injections can be used to achieve the desireddose. In certain embodiments, the pharmaceutical agent is administeredby infusion over an extended period of time or continuously. Doses canbe stated as the amount of pharmaceutical agent per hour, day, week, ormonth. Doses can also be stated as mg/kg or g/kg.

As used herein, “effective amount” or “therapeutically effective amount”means the amount of active pharmaceutical agent sufficient to effectuatea desired physiological outcome in an individual in need of the agent.The effective amount can vary among individuals depending on the healthand physical condition of the individual to be treated, the taxonomicgroup of the individuals to be treated, the formulation of thecomposition, assessment of the individual's medical condition, and otherrelevant factors.

As used herein, “Fredrickson Type I” is also known as “Lipoproteinlipase deficiency”, “LPLD”, “Familial Chylomicronemia Syndrome” or “FCS”and exists in several forms: Type 1a (also known as Buerger-Gruestzsyndrome) is a lipoprotein lipase deficiency commonly due to adeficiency of LPL or altered ApoC-II; Type Ib (also known as familialapoprotein CII deficiency) is a condition caused by lack of lipoproteinlipase activator apoprotein C-II; and Type Ic is a chylomicronemia dueto circulating inhibitor of lipoprotein lipase. Type I is a raredisorder that usually presents in childhood. It is characterized bysevere elevations in chylomicrons and extremely elevated TG levels(always reaching well above 1000 mg/dL and not infrequently rising ashigh as 10,000 mg/dL or more) with episodes of abdominal pain, recurrentacute pancreatitis, eruptive cutaneous xanthomata, andhepatosplenomegaly. Patients rarely develop atherosclerosis, perhapsbecause their plasma lipoprotein particles are too large to enter intothe arterial intima (Nordestgaard et al., J Lipid Res, 1988,29:1491-1500; Nordestgaard et al., Arteriosclerosis, 1988, 8:421-428).Type I is usually caused by mutations of either the LPL gene, or of thegene's cofactor ApoC-II, resulting in the inability of affectedindividuals to produce sufficient functionally active LPL. Patients areeither homozygous for such mutations or compound heterozygous.Fredrickson Type I can also be due to mutations in the GPIHBP1, APOA5,LMF1 or other genes leading to dysfunctional LPL. Brunzell, In: Pagon RA, Adam M P, Bird T D, Dolan C R, Fong C T, Stephens K, editors.GeneReviews™ [Internet]. Seattle (Wash.): University of Washington,Seattle; 1993-2013. 1999 Oct. 12 [updated 2011 Dec. 15]. Further,Fredrickson Type I, in some instances, can be due to the presence of LPLinhibitors (e.g., anti-LPL antibodies) in an individual causingdysfunctional LPL. The prevalence of Fredrickson Type I is approximately1 in 1,000,000 in the general population and much higher in South Africaand Eastern Quebec as a result of a founder effect. Patients respondminimally, or not at all, to TG-lowering drugs (Tremblay et al., J ClinLipidol, 2011, 5:37-44; Brisson et al., Pharmacogenet Genom, 2010,20:742-747) and hence restriction of dietary fat to 20 grams/day or lessis used to manage the symptoms of this rare disorder. As used herein,“fully complementary” or “100% complementary” means each nucleobase of anucleobase sequence of a first nucleic acid has a complementarynucleobase in a second nucleobase sequence of a second nucleic acid. Incertain embodiments, a first nucleic acid is an antisense compound and asecond nucleic acid is a target nucleic acid.

As used herein, “glucose” is a monosaccharide used by cells as a sourceof energy and inflammatory intermediate. “Plasma glucose” refers toglucose present in the plasma.

As used herein, “high density lipoprotein-C” or “HDL-C” meanscholesterol associated with high density lipoprotein particles.Concentration of HDL-C in serum (or plasma) is typically quantified inmg/dL or nmol/L. “Serum HDL-C” and “plasma HDL-C” mean HDL-C in serumand plasma, respectively.

As used herein, “HMG-CoA reductase inhibitor” means an agent that actsthrough the inhibition of the enzyme HMG-CoA reductase, such asatorvastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, andsimvastatin.

As used herein, “hypercholesterolemia” means a condition characterizedby elevated cholesterol or circulating (plasma) cholesterol,LDL-cholesterol and VLDL-cholesterol, as per the guidelines of theExpert Panel Report of the National Cholesterol Educational Program(NCEP) of Detection, Evaluation of Treatment of high cholesterol inadults (see, Arch. Int. Med. (1988) 148, 36-39).

As used herein, “hyperlipidemia” or “hyperlipemia” is a conditioncharacterized by elevated serum lipids or circulating (plasma) lipids.This condition manifests an abnormally high concentration of fats. Thelipid fractions in the circulating blood are cholesterol, low densitylipoproteins, very low density lipoproteins, chylomicrons andtriglycerides. The Fredrickson classification of hyperlipidemias isbased on the pattern of TG and cholesterol-rich lipoprotein particles,as measured by electrophoresis or ultracentrifugation and is commonlyused to characterize primary causes of hyperlipidemias such ashypertriglyceridemia (Fredrickson and Lee, Circulation, 1965,31:321-327; Fredrickson et al., New Eng J Med, 1967, 276 (1): 34-42).

As used herein, “hypertriglyceridemia” means a condition characterizedby elevated triglyceride levels. Hypertriglyceridemia is the consequenceof increased production and/or reduced or delayed catabolism oftriglyceride (TG)-rich lipoproteins: VLDL and, to a lesser extent,chylomicrons (CM). Its etiology includes primary (i.e. genetic causes)and secondary (other underlying causes such as diabetes, metabolicsyndrome/insulin resistance, obesity, physical inactivity, cigarettesmoking, excess alcohol and a diet very high in carbohydrates) factorsor, most often, a combination of both (Yuan et al. CMAJ, 2007,176:1113-1120). Hypertriglyceridemia is a common clinical traitassociated with an increased risk of cardiometabolic disease (Hegele etal. 2009, Hum Mol Genet, 18: 4189-4194; Hegele and Pollex 2009, Mol CellBiochem, 326: 35-43) as well as of occurrence of acute pancreatitis inthe most severe forms (Toskes 1990, Gastroenterol Clin North Am, 19:783-791; Gaudet et al. 2010, Atherosclerosis Supplements, 11: 55-60;Catapano et al. 2011, Atherosclerosis, 217S: S1-S44; Tremblay et al.2011, J Clin Lipidol, 5: 37-44). Examples of cardiometabolic diseaseinclude, but are not limited to, diabetes, metabolic syndrome/insulinresistance, and genetic disorders such as familial chylomicronemiasyndrome (FCS), familial combined hyperlipidemia and familialhypertriglyceridemia. Borderline high TG levels (150-199 mg/dL) arecommonly found in the general population and are a common component ofthe metabolic syndrome/insulin resistance states. The same is true forhigh TG levels (200-499 mg/dL) except that as plasma TG levels increase,underlying genetic factors play an increasingly important etiologicrole. Very high TG levels (≥500 mg/dL) are most often associated withelevated CM levels as well, and are accompanied by increasing risk foracute pancreatitis. The risk of pancreatitis is considered clinicallysignificant if TG levels exceed 880 mg/dL (>10 mmol) and the EuropeanAtherosclerosis Society/European Society of Cardiology (EAS/ESC) 2011guidelines state that actions to prevent acute pancreatitis aremandatory (Catapano et al. 2011, Atherosclerosis, 217S: S1-S44).According to the EAS/ESC 2011 guidelines, hypertriglyceridemia is thecause of approximately 10% of all cases of pancreatitis, and developmentof pancreatitis can occur at TG levels between 440-880 mg/dL. Based onevidence from clinical studies demonstrating that elevated TG levels arean independent risk factor for atherosclerotic CVD, the guidelines fromboth the National Cholesterol Education Program Adult Treatment PanelIII (NCEP 2002, Circulation, 106: 3143-421) and the American DiabetesAssociation (ADA 2008, Diabetes Care, 31: S12-S54.) recommend a targetTG level of less than 150 mg/dL to reduce cardiovascular risk.

As used herein, “identifying” or “selecting an animal with metabolic orcardiovascular disease” means identifying or selecting a subject proneto or having been diagnosed with a metabolic disease, a cardiovasculardisease, or a metabolic syndrome; or, identifying or selecting a subjecthaving any symptom of a metabolic disease, cardiovascular disease, ormetabolic syndrome including, but not limited to, hypercholesterolemia,hyperglycemia, hyperlipidemia, hypertriglyceridemia, hypertensionincreased insulin resistance, decreased insulin sensitivity, abovenormal body weight, and/or above normal body fat content or anycombination thereof. Such identification can be accomplished by anymethod, including but not limited to, standard clinical tests orassessments, such as measuring serum or circulating (plasma)cholesterol, measuring serum or circulating (plasma) blood-glucose,measuring serum or circulating (plasma) triglycerides, measuringblood-pressure, measuring body fat content, measuring body weight, andthe like.

As used herein, “improved cardiovascular outcome” means a reduction inthe occurrence of adverse cardiovascular events, or the risk thereof.Examples of adverse cardiovascular events include, without limitation,death, reinfarction, stroke, cardiogenic shock, pulmonary edema, cardiacarrest, and atrial dysrhythmias.

As used herein, “immediately adjacent” means there are no interveningelements between the immediately adjacent elements, for example, betweenregions, segments, nucleotides and/or nucleosides.

As used herein, “increasing HDL” or “raising HDL” means increasing thelevel of HDL in an animal after administration of at least one compoundof the invention, compared to the HDL level in an animal notadministered any compound.

As used herein, “individual” or “subject” or “animal” means a human ornon-human animal selected for treatment or therapy.

As used herein, “individual in need thereof” refers to a human ornon-human animal selected for treatment or therapy that is in need ofsuch treatment or therapy.

As used herein, “induce”, “inhibit”, “potentiate”, “elevate”,“increase”, “decrease”, “reduce” or the like denote quantitativedifferences between two states. For example, “an amount effective toinhibit the activity or expression of apoCIII” means that the level ofactivity or expression of apoCIII in a treated sample will differ fromthe level of apoCIII activity or expression in an untreated sample. Suchterms are applied to, for example, levels of expression, and levels ofactivity.

As used herein, “inflammatory condition” refers to a disease, diseasestate, syndrome, or other condition resulting in inflammation. Forexample, rheumatoid arthritis and liver fibrosis are inflammatoryconditions. Other examples of inflammatory conditions include sepsis,myocardial ischemia/reperfusion injury, adult respiratory distresssyndrome, nephritis, graft rejection, inflammatory bowel disease,multiple sclerosis, arteriosclerosis, atherosclerosis and vasculitis.

As used herein, “inhibiting the expression or activity” refers to areduction or blockade of the expression or activity of a RNA or proteinand does not necessarily indicate a total elimination of expression oractivity.

As used herein, “insulin resistance” is defined as the condition inwhich normal amounts of insulin are inadequate to produce a normalinsulin response from fat, muscle and liver cells. Insulin resistance infat cells results in hydrolysis of stored triglycerides, which elevatesfree fatty acids in the blood plasma. Insulin resistance in musclereduces glucose uptake whereas insulin resistance in liver reducesglucose storage, with both effects serving to elevate blood glucose.High plasma levels of insulin and glucose due to insulin resistanceoften leads to metabolic syndrome and type 2 diabetes.

As used herein, “insulin sensitivity” is a measure of how effectively anindividual processes glucose. An individual having high insulinsensitivity effectively processes glucose whereas an individual with lowinsulin sensitivity does not effectively process glucose.

As used herein, “lipid-lowering” means a reduction in one or more lipids(e.g., LDL, VLDL) in a subject. “Lipid-raising” means an increase in alipid (e.g., HDL) in a subject. Lipid-lowering or lipid-raising canoccur with one or more doses over time.

As used herein, “lipid-lowering therapy” or “lipid lowering agent” meansa therapeutic regimen provided to a subject to reduce one or more lipidsin a subject. In certain embodiments, a lipid-lowering therapy isprovided to reduce one or more of apo(a), apoCIII, CETP, apoB, totalcholesterol, LDL-C, VLDL-C, IDL-C, non-HDL-C, triglycerides, small denseLDL particles, and Lp(a) in a subject. Examples of lipid-loweringtherapy include, but are not limited to, apoB inhibitors, statins,fibrates and MTP inhibitors.

As used herein, “lipoprotein”, such as VLDL, LDL and HDL, refers to agroup of proteins found in the serum, plasma and lymph and are importantfor lipid transport. The chemical composition of each lipoproteindiffers, for example, in that the HDL has a higher proportion of proteinversus lipid, whereas the VLDL has a lower proportion of protein versuslipid.

As used herein, “Lipoprotein Lipase” or “LPL” refers to an enzyme thathydrolyzes TGs found in lipoproteins, such as CM or VLDL, into freefatty acids and monoacylglycerols. LPL requires apo C-II as a cofactorto function in hydrolyzing TGs. LPL is mainly produced in skeletalmuscle, fat tissue, and heart muscle. Hydrolysis and removal of TG fromCM and VLDL normally protects against excessive postprandial rise in CMmass and TG.

As used herein, “Lipoprotein lipase deficient”, “lipoprotein lipasedeficiency”, “LPL deficiency” or “LPLD” is also known as “Fredrickson'sType I dyslipidemia”, “chylomicronemia”, “Familial ChylomicronemiaSyndrome” or “FCS”. Although subjects with LPLD generally lack LPL orLPL activity necessary for effective breakdown of fatty acids such asTGs, these subjects may still have a minimal LPL activity or express aminimal level of LPL. In some instances, a LPLD subject may express LPLor have LPL activity up to about, or no more than, 20%, 19%, 18%, 17%,16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%activity. In other instances, the LPLD subject has no measurable LPL orLPL activity. One embodiment of LPLD encompasses subjects with“hyperlipoproteinemia type Ia” (also known as “Fredrickson's Type Ia”)and refers to the inability of the subjects to produce sufficientfunctional lipoprotein lipase enzymes necessary for effective breakdownof fatty acids such as TGs. The inability to breakdown TGs leads tohypertriglyceridemia in the subject and, often more than 12 hours aftermeals, hyperTG and chylomicronemia are still present and visible aslipemia. Type Ia is commonly caused by one or more mutations in the LPLgene. As disclosed herein, LPLD also encompasses subjects that havedysfunctional lipoprotein lipase such as those subjects with“hyperlipoproteinemia type Ib” (also known as “Fredrickson's Type Ib”)and “hyperlipoproteinemia type Ic” (also known as “Fredrickson's TypeIc”). Type Ib is caused by lack of lipoprotein lipase activatorapoprotein C-II. Type Ic is due to a circulating inhibitor oflipoprotein lipase. As with Type Ia, Type Ib/Ic subjects suffer from aninability to breakdown TGs leading to hypertriglyceridemia and hyperTGand chylomicronemia are still present and visible as lipemia often morethan 12 hours after meals. In certain embodiments, LPLD is associatedwith at least one mutation in the LPL gene such as P207L, G188L or D9Nor other mutations that affect LPL (Brunzell, In: Pagon R A, Adam M P,Bird T D, Dolan C R, Fong C T, Stephens K, editors. GeneReviews™[Internet]. Seattle (Wash.): University of Washington, Seattle;1993-2013. 1999 Oct. 12 [updated 2011 Dec. 15]).

As used herein, “low density lipoprotein-cholesterol (LDL-C)” meanscholesterol carried in low density lipoprotein particles. Concentrationof LDL-C in serum (or plasma) is typically quantified in mg/dL ornmol/L. “Serum LDL-C” and “plasma LDL-C” mean LDL-C in the serum andplasma, respectively.

As used herein, “major risk factors” refers to factors that contributeto a high risk for a particular disease or condition. In certainembodiments, major risk factors for coronary heart disease include,without limitation, cigarette smoking, hypertension, high LDL, lowHDL-C, family history of coronary heart disease, age, and other factorsdisclosed herein.

As used herein, “metabolic disorder” or “metabolic disease” refers to acondition characterized by an alteration or disturbance in metabolicfunction. “Metabolic” and “metabolism” are terms well known in the artand generally include the whole range of biochemical processes thatoccur within a living organism. Metabolic disorders include, but are notlimited to, hyperglycemia, prediabetes, diabetes (type 1 and type 2),obesity, insulin resistance, metabolic syndrome and dyslipidemia due totype 2 diabetes.

As used herein, “metabolic syndrome” means a condition characterized bya clustering of lipid and non-lipid cardiovascular risk factors ofmetabolic origin. In certain embodiments, metabolic syndrome isidentified by the presence of any 3 of the following factors: waistcircumference of greater than 102 cm in men or greater than 88 cm inwomen; serum triglyceride of at least 150 mg/dL; HDL-C less than 40mg/dL in men or less than 50 mg/dL in women; blood pressure of at least130/85 mmHg; and fasting glucose of at least 110 mg/dL. Thesedeterminants can be readily measured in clinical practice (JAMA, 2001,285: 2486-2497).

“Parenteral administration” means administration through injection orinfusion. Parenteral administration includes subcutaneousadministration, intravenous administration, intramuscularadministration, intraarterial administration, intraperitonealadministration, or intracranial administration, e.g. intrathecal orintracerebroventricular administration. Administration can becontinuous, chronic, short or intermittent.

As used herein, “peptide” means a molecule formed by linking at leasttwo amino acids by amide bonds. Peptide refers to polypeptides andproteins.

As used herein, “pharmaceutical agent” means a substance that provides atherapeutic benefit when administered to an individual. For example, incertain embodiments, an antisense oligonucleotide targeted to apoCIII isa pharmaceutical agent.

As used herein, “pharmaceutical composition” or “composition” means amixture of substances suitable for administering to an individual. Forexample, a pharmaceutical composition can comprise one or more activeagents and a pharmaceutical carrier e.g., a sterile aqueous solution.

As used herein, “pharmaceutically acceptable derivative” encompassesderivatives of the compounds described herein such as solvates,hydrates, esters, prodrugs, polymorphs, isomers, isotopically labelledvariants, pharmaceutically acceptable salts and other derivatives knownin the art.

As used herein, “pharmaceutically acceptable salts” meansphysiologically and pharmaceutically acceptable salts of antisensecompounds, i.e., salts that retain the desired biological activity ofthe parent compound and do not impart undesired toxicological effectsthereto. The term “pharmaceutically acceptable salt” or “salt” includesa salt prepared from pharmaceutically acceptable non-toxic acids orbases, including inorganic or organic acids and bases. “Pharmaceuticallyacceptable salts” of the compounds described herein may be prepared bymethods well-known in the art. For a review of pharmaceuticallyacceptable salts, see Stahl and Wermuth, Handbook of PharmaceuticalSalts: Properties, Selection and Use (Wiley-VCH, Weinheim, Germany,2002). Sodium salts of antisense oligonucleotides are useful and arewell accepted for therapeutic administration to humans. Accordingly, inone embodiment the compounds described herein are in the form of asodium salt.

As used herein, “portion” means a defined number of contiguous (i.e.linked) nucleobases of a nucleic acid. In certain embodiments, a portionis a defined number of contiguous nucleobases of a target nucleic acid.In certain embodiments, a portion is a defined number of contiguousnucleobases of an antisense compound.

As used herein, “prevent” or “preventing” refers to delaying orforestalling the onset or development of a disease, disorder, orcondition for a period of time from minutes to indefinitely. Preventalso means reducing risk of developing a disease, disorder, orcondition.

As used herein, “raise” means to increase in amount. For example, toraise plasma HDL levels means to increase the amount of HDL in theplasma.

As used herein, “reduce” means to bring down to a smaller extent, size,amount, or number. For example, to reduce plasma triglyceride levelsmeans to bring down the amount of triglyceride in the plasma.

As used herein, “region” or “target region” is defined as a portion ofthe target nucleic acid having at least one identifiable structure,function, or characteristic. For example, a target region may encompassa 3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, acoding region, a translation initiation region, translation terminationregion, or other defined nucleic acid region. The structurally definedregions for apoCIII can be obtained by accession number from sequencedatabases such as NCBI and such information is incorporated herein byreference. In certain embodiments, a target region may encompass thesequence from a 5′ target site of one target segment within the targetregion to a 3′ target site of another target segment within the targetregion.

As used herein, “second agent” or “second therapeutic agent” means anagent that can be used in combination with a “first agent”. A secondtherapeutic agent can include, but is not limited to, antisenseoligonucleotides targeting apoCIII. A second agent can also includeanti-apoCIII antibodies, apoCIII peptide inhibitors, cholesterollowering agents, lipid lowering agents, glucose lowering agents andanti-inflammatory agents.

As used herein, “segments” are defined as smaller, sub-portions ofregions within a nucleic acid. For example, a “target segment” means thesequence of nucleotides of a target nucleic acid to which one or moreantisense compounds is targeted. “5′ target site” refers to the 5′-mostnucleotide of a target segment. “3′ target site” refers to the 3′-mostnucleotide of a target segment. Alternatively, a “start site” can referto the 5′-most nucleotide of a target segment and a “stop site” refersto the 3′-most nucleotide of a target segment. A target segment can alsobegin at the “start site” of one sequence and end at the “stop site” ofanother sequence.

As used herein, “statin” means an agent that inhibits the activity ofHMG-CoA reductase.

As used herein, “subcutaneous administration” means administration justbelow the skin.

As used herein, “subject” means a human or non-human animal selected fortreatment or therapy.

As used herein, “symptom of cardiovascular disease or disorder” means aphenomenon that arises from and accompanies the cardiovascular diseaseor disorder and serves as an indication of it. For example, angina;chest pain; shortness of breath; palpitations; weakness; dizziness;nausea; sweating; tachycardia; bradycardia; arrhythmia; atrialfibrillation; swelling in the lower extremities; cyanosis; fatigue;fainting; numbness of the face; numbness of the limbs; claudication orcramping of muscles; bloating of the abdomen; or fever are symptoms ofcardiovascular disease or disorder.

As used herein, “targeting” or “targeted” means the process of designand selection of an antisense compound that will specifically hybridizeto a target nucleic acid and induce a desired effect.

As used herein, “therapeutically effective amount” means an amount of apharmaceutical agent that provides a therapeutic benefit to anindividual.

As used herein, “therapeutic lifestyle change” means dietary andlifestyle changes intended to lower fat/adipose tissue mass and/orcholesterol. Such change can reduce the risk of developing heartdisease, and may includes recommendations for dietary intake of totaldaily calories, total fat, saturated fat, polyunsaturated fat,monounsaturated fat, carbohydrate, protein, cholesterol, insolublefiber, as well as recommendations for physical activity.

As used herein, “treat” or “treating” refers to administering a compounddescribed herein to effect an alteration or improvement of a disease,disorder, or condition.

As used herein, “triglyceride” or “TG” means a lipid or neutral fatconsisting of glycerol combined with three fatty acid molecules.

As used herein, “type 2 diabetes,” (also known as “type 2 diabetesmellitus”, “diabetes mellitus, type 2”, “non-insulin-dependentdiabetes”, “NIDDM”, “obesity related diabetes”, or “adult-onsetdiabetes”) is a metabolic disorder that is primarily characterized byinsulin resistance, relative insulin deficiency, and hyperglycemia.

Certain Embodiments

Certain embodiments provide a compounds and methods for decreasingApoCIII mRNA and protein expression. In certain embodiments, thecompound is an ApoCIII specific inhibitor for treating, preventing, orameliorating an ApoCIII associated disease. In certain embodiments, thecompound is an antisense oligonucleotide targeting ApoCIII. In certainembodiments, the compound is an modified oligonucleotide targetingApoCIII and a conjugate group.

In certain embodiments, a compound comprises a siRNA or antisenseoligonucleotide targeted to Apolipoprotein C-III (ApoC-III) known in theart and a conjugate group described herein. Examples of antisenseoligonucleotides targeted to ApoC-III suitable for conjugation includebut are not limited to those disclosed in US Patent ApplicationPublication No. US 2013/0317085, which is incorporated by reference inits entirety herein. In certain embodiments, a compound comprises anantisense oligonucleotide having a nucleobase sequence of any of SEQ IDNOs 19-96 and 209-221 disclosed in US 2013/0317085 and a conjugate groupdescribed herein. The nucleobase sequences of all of the aforementionedreferenced SEQ ID NOs are incorporated by reference herein.

In certain embodiments, the modified oligonucleotide with the conjugategroup has a nucleobase sequence comprising at least 8 contiguousnucleobases of a sequence selected from any sequence disclosed in U.S.Pat. No. 7,598,227, U.S. Pat. No. 7,750,141, PCT Publication WO2004/093783 or PCT Publication WO 2012/149495, allincorporated-by-reference herein. In certain embodiments, the modifiedoligonucleotide has a sequence selected from any sequence disclosed inU.S. Pat. No. 7,598,227, U.S. Pat. No. 7,750,141, PCT Publication WO2004/093783 or PCT Publication WO 2012/149495, allincorporated-by-reference herein.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting ApoCIII and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides. Incertain embodiments, the modified oligonucleotide with the conjugategroup consists of 15 to 30, 18 to 24, 19 to 22, 13 to 25, 14 to 25, 15to 25 linked nucleosides. In certain embodiments, the modifiedoligonucleotide with the conjugate group comprises at least 12, at least13, at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24, at least 25, at least 26, at least 27, at least 28, at least 29 or30 linked nucleosides. In certain embodiments, the modifiedoligonucleotide with the conjugate group consists of 20 linkednucleosides.

Certain embodiments provide a compound comprising a modifiedoligonucleotide with a conjugate group targeting ApoCIII and has asequence complementary to any of the sequences set forth in GENBANKAccession No. NM_000040.1 (incorporated herein as SEQ ID NO: 1), GENBANKAccession No. NT_033899.8 truncated from nucleotides 20262640 to20266603 (incorporated herein as SEQ ID NO: 2), and/or GenBank AccessionNo. NT_035088.1 truncated from nucleotides 6238608 to U.S. Pat. No.6,242,565 (incorporated herein as SEQ ID NO: 3). In certain embodiments,the modified oligonucleotide is at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98% or at least100% complementary to any of SEQ ID NOs: 1-3. In certain embodiments,the compound comprises a modified oligonucleotide targeting ApoCIII anda conjugate group, wherein the modified oligonucleotide comprises atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, or 20 contiguous nucleobases complementary to an equal lengthportion of any of SEQ ID NOs: 1-3. In certain embodiments, the compoundcomprises a modified oligonucleotide targeting an ApoCIII segment and aconjugate group, wherein the modified oligonucleotide comprises at least8, at least 9, at least 10, at least 11, at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least19, or 20 contiguous nucleobases complementary to an equal lengthportion of any of the target segments shown in Tables 121 and 124. Inthe tables, the “Start Site” refers to the 5′-most nucleotide of atarget segment and “Stop Site” refers to the 3′-most nucleotide of atarget segment. A target segment can range from the start site to thestop site of each sequence listed in the tables. Alternatively, thetarget segment can range from the start site of one sequence and end atthe stop site of another sequence. For example, as shown in the tables,a target segment can range from 3533 to 3552, the start site to the stopsite of SEQ ID NO: 87. In another example, as shown in the tables, atarget segment can range from 3514 to 3558, the start site of SEQ ID NO:83 to the stop site of SEQ ID NO: 88. In certain embodiments, theantisense compound comprises at least 8 nucleobases of the sequence ofSEQ ID NO: 87. In certain embodiments, the antisense compound comprisesthe sequence of SEQ ID NO: 87. In certain embodiments, the antisensecompound consists of the sequence of SEQ ID NO: 87. In certainembodiments, the antisense compound is ISIS 304801.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting ApoCIII and a conjugate group, wherein thenucleobase sequence of the modified oligonucleotide is at least 80%, atleast 85%, at least 90%, at least 95%, or 100% complementary to any ofSEQ ID NOs: 1-3. Certain embodiments provide a compound comprising amodified oligonucleotide targeting ApoCIII and a conjugate group,wherein the nucleobase sequence of the modified oligonucleotide is atleast 80%, at least 85%, at least 90%, at least 95%, or 100%complementary to any of the target segments disclosed herein.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting ApoCIII and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andcomprises a nucleobase sequence comprising a portion of at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, or20 contiguous nucleobases complementary to an equal length portion ofnucleobases 3533 to 3552 of SEQ ID NO: 3, wherein the nucleobasesequence of the modified oligonucleotide is at least 80% complementaryto SEQ ID NO: 3.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting ApoCIII and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andcomprises a nucleobase sequence comprising at least 8, at least 9, atleast 10, at least 11, at least 12, at least 13, at least 14, at least15, at least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, at least 22, at least 23, at least 24, at least 25, at least26, at least 27, at least 28, at least 29 or 30 contiguous nucleobasescomplementary to an equal length portion of nucleobases 3514 to 3558 ofSEQ ID NO: 3, wherein the nucleobase sequence of the modifiedoligonucleotide is at least 80% complementary to SEQ ID NO: 3. Certainembodiments provide a compound comprising a modified oligonucleotidetargeting ApoCIII and a conjugate group, wherein the modifiedoligonucleotide consists of 12 to 30 linked nucleosides and has anucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or 20 contiguous nucleobasesof any of the nucleobase sequences of SEQ ID NOs: 19-96, 209-221. Incertain embodiments, the conjugated modified oligonucleotide has anucleobase sequence comprising at least 8 contiguous nucleobases of anyone of the nucleobase sequences of SEQ ID NOs: 19-96, 209-221. Incertain embodiments, the compound consists of any one of SEQ ID NOs:19-96, 209-221 and a conjugate group.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting ApoCIII and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides and hasa nucleobase sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or 20 contiguous nucleobasesof the nucleobase sequence of SEQ ID NO: 87. In certain embodiments, themodified oligonucleotide with the conjugate group has a nucleobasesequence comprising at least 8 contiguous nucleobases of the nucleobasesequence of SEQ ID NO: 87. In certain embodiments, the compound consistsof SEQ ID NO: 87 and a conjugate group.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises a modified oligonucleotidewith ISIS 304801 with a 5′-X, wherein X is a conjugate group comprisingGalNAc. In certain embodiments, the antisense compound consists of amodified oligonucleotide with ISIS 304801 with a 5′-X, wherein X is aconjugate group comprising GalNAc.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises the conjugated modifiedoligonucleotide ISIS 678354. In certain embodiments, the antisensecompound consists of the conjugated modified oligonucleotide ISIS678354.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises the conjugated modifiedoligonucleotide ISIS 678357. In certain embodiments, the antisensecompound consists of the conjugated modified oligonucleotide ISIS678357.

In certain embodiments, the present disclosure provides conjugatedantisense compounds represented by the following structure. In certainembodiments, the antisense compound comprises a modified oligonucleotidewith the nucleobase sequence of SEQ ID NO: 87 with a 5′-GalNAc withvariability in the sugar mods of the wings. In certain embodiments, theantisense compound consists of a modified oligonucleotide with thenucleobase sequence of SEQ ID NO: 87 with a 5′-GalNAc with variabilityin the sugar mods of the wings.

Wherein either R¹ is —OCH₂CH₂OCH₃ (MOE) and R² is H; or R¹ and R²together form a bridge, wherein R¹ is —O— and R² is —CH₂—, —CH(CH₃)—, or—CH₂CH₂—, and R¹ and R² are directly connected such that the resultingbridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and —O—CH₂CH₂—;

And for each pair of R³ and R⁴ on the same ring, independently for eachring: either R³ is selected from H and —OCH₂CH₂OCH₃ and R⁴ is H; or R³and R⁴ together form a bridge, wherein R³ is —O—, and R⁴ is —CH₂—,—CH(CH₃)—, or —CH₂CH₂— and R³ and R⁴ are directly connected such thatthe resulting bridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and—O—CH₂CH₂—;

And R⁵ is selected from H and —CH₃;

And Z is selected from S⁻ and O⁻.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting ApoCIII and a conjugate group, wherein themodified oligonucleotide is single-stranded.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting ApoCIII and a conjugate group, wherein atleast one internucleoside linkage is a modified internucleoside linkage.In certain embodiments, the modified internucleoside linkage is aphosphorothioate internucleoside linkage. In certain embodiments, atleast 1, at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9 or at least 10 internucleoside linkagesof said modified oligonucleotide are phosphorothioate internucleosidelinkages. In certain embodiments, each internucleoside linkage is aphosphorothioate internucleoside linkage. In certain embodiments, themodified oligonucleotide comprises at least 1, at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9or at least 10 phosphodiester internucleoside linkages. In certainembodiments, each internucleoside linkage of the modifiedoligonucleotide is selected from a phosphodiester internucleosidelinkage and a phosphorothioate internucleoside linkage.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting ApoCIII and a conjugate group, wherein atleast one nucleoside comprises a modified nucleobase. In certainembodiments, the modified nucleobase is a 5-methylcytosine.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting ApoCIII and a conjugate group, wherein themodified oligonucleotide comprises at least one modified sugar. Incertain embodiments, the modified sugar is a bicyclic sugar. In certainembodiments, the modified sugar comprises a 2′-O-methoxyethyl, aconstrained ethyl, a 3′-fluoro-HNA or a 4′-(CH₂)_(n)—O-2′ bridge,wherein n is 1 or 2.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting ApoCIII and a conjugate group, wherein themodified oligonucleotide consists of 12 to 30 linked nucleosides andcomprises: (a) a gap segment consisting of linked deoxynucleosides; (b)a 5′ wing segment consisting of linked nucleosides; (c) a 3′ wingsegment consisting of linked nucleosides; and wherein the gap segment ispositioned between the 5′ wing segment and the 3′ wing segment andwherein each nucleoside of each wing segment comprises a modified sugar.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting ApoCIII and a conjugate group, wherein themodified oligonucleotide consists of 20 linked nucleosides andcomprises: (a) a gap segment consisting of ten linked deoxynucleosides;(b) a 5′ wing segment consisting of five linked nucleosides; (c) a 3′wing segment consisting of five linked nucleosides; and wherein the gapsegment is positioned between the 5′ wing segment and the 3′ wingsegment, wherein each nucleoside of each wing segment comprises a2′-O-methoxyethyl sugar, wherein at least one internucleoside linkage isa phosphorothioate linkage and wherein each cytosine residue is a5-methylcytosine.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting ApoCIII and a conjugate group, wherein themodified oligonucleotide consists of 20 linked nucleosides and has anucleobase sequence comprising at least 8 contiguous nucleobases of anyof SEQ ID NOs: 19-96, 209-221, wherein the modified oligonucleotidecomprises: (a) a gap segment consisting of ten linked deoxynucleosides;(b) a 5′ wing segment consisting of five linked nucleosides; (c) a 3′wing segment consisting of five linked nucleosides; and wherein the gapsegment is positioned between the 5′ wing segment and the 3′ wingsegment, wherein each nucleoside of each wing segment comprises a2′-O-methoxyethyl sugar, wherein at least one internucleoside linkage isa phosphorothioate linkage and wherein each cytosine residue is a5-methylcytosine.

Certain embodiments provide a compound comprising a modifiedoligonucleotide targeting ApoCIII and a conjugate group, wherein themodified oligonucleotide consists of 20 linked nucleosides and has anucleobase sequence comprising at least 8 contiguous nucleobases of SEQID NO: 87, wherein the modified oligonucleotide comprises: (a) a gapsegment consisting of ten linked deoxynucleosides; (b) a 5′ wing segmentconsisting of five linked nucleosides; (c) a 3′ wing segment consistingof five linked nucleosides; and wherein the gap segment is positionedbetween the 5′ wing segment and the 3′ wing segment, wherein eachnucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar,wherein at least one internucleoside linkage is a phosphorothioatelinkage and wherein each cytosine residue is a 5-methylcytosine.

Certain embodiments provide a modified oligonucleotide targeting ApoCIIIand a conjugate group, wherein the modified oligonucleotide consists of20 linked nucleosides with the nucleobase sequence of SEQ ID NO: 87,wherein the modified oligonucleotide comprises: (a) a gap segmentconsisting of ten linked deoxynucleosides; (b) a 5′ wing segmentconsisting of five linked nucleosides; (c) a 3′ wing segment consistingof five linked nucleosides; and wherein the gap segment is positionedbetween the 5′ wing segment and the 3′ wing segment, wherein eachnucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar,wherein at least one internucleoside linkage is a phosphorothioatelinkage and wherein each cytosine residue is a 5-methylcytosine.

In certain embodiments, the conjugate group is linked to the modifiedoligonucleotide at the 5′ end of the modified oligonucleotide. Incertain embodiments, the conjugate group is linked to the modifiedoligonucleotide at the 3′ end of the modified oligonucleotide.

In certain embodiments, the conjugate group comprises exactly oneligand. In certain embodiments, the conjugate group comprises one ormore ligands. In certain embodiments, the conjugate group comprisesexactly two ligands. In certain embodiments, the conjugate groupcomprises two or more ligands. In certain embodiments, the conjugategroup comprises three or more ligands. In certain embodiments, theconjugate group comprises exactly three ligands. In certain embodiments,each ligand is selected from among: a polysaccharide, modifiedpolysaccharide, mannose, galactose, a mannose derivative, a galactosederivative, D-mannopyranose, L-Mannopyranose, D-Arabinose, L-Galactose,D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-Galactose,L-Galactose, α-D-Mannofuranose, β-D-Mannofuranose, α-D-Mannopyranose,β-D-Mannopyranose, α-D-Glucopyranose, β-D-Glucopyranose,α-D-Glucofuranose, β-D-Glucofuranose, α-D-fructofuranose,α-D-fructopyranose, α-D-Galactopyranose, β-D-Galactopyranose,α-D-Galactofuranose, β-D-Galactofuranose, glucosamine, sialic acid,α-D-galactosamine, N-Acetylgalactosamine,2-Amino-3-O—[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose,2-Deoxy-2-methylamino-L-glucopyranose,4,6-Dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose,2-Deoxy-2-sulfoamino-D-glucopyranose, N-Glycoloyl-α-neuraminic acid,5-thio-β-D-glucopyranose, methyl2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside,4-Thio-β-D-galactopyranose, ethyl3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside,2,5-Anhydro-D-allononitrile, ribose, D-ribose, D-4-thioribose, L-ribose,L-4-thioribose. In certain embodiments, each ligand is N-acetylgalactosamine.

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises at least onephosphorus linking group or neutral linking group.

In certain embodiments, the conjugate group comprises a structureselected from among:

wherein n is from 1 to 12; and

wherein m is from 1 to 12.

In certain embodiments, the conjugate group has a tether having astructure selected from among:

-   -   wherein L is either a phosphorus linking group or a neutral        linking group;    -   Z₁ is C(═O)O—R₂;    -   Z₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky;    -   R₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky; and    -   each m₁ is, independently, from 0 to 20 wherein at least one m₁        is greater than 0 for each tether.        In certain embodiments, the conjugate group has a tether having        a structure selected from among:

-   -   wherein Z₂ is H or CH₃; and    -   each m₁ is, independently, from 0 to 20 wherein at least one m₁        is greater than 0 for each tether.        In certain embodiments, the conjugate group has tether having a        structure selected from among:

wherein n is from 1 to 12; and

wherein m is from 1 to 12.

In certain embodiments, the conjugate group is covalently attached tothe modified oligonucleotide.In certain embodiments, the compound has a structure represented by theformula:

A-B-C-DE-F)_(q)

wherein

A is the modified oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by theformula:

AB_(n) ₂ C_(n) ₁ D_(n) ₃ E-F)_(q)

wherein:

A is the modified oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand;

each n is independently 0 or 1; and

q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by theformula:

A-B-CE-F)_(q)

wherein

A is the modified oligonucleotide;

B is the cleavable moiety;

C is the conjugate linker;

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by theformula:

A-C-DE-F)_(q)

wherein

A is the modified oligonucleotide;

C is the conjugate linker;

D is the branching group;

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by theformula:

A-CE-F)_(q)

wherein

A is the modified oligonucleotide;

C is the conjugate linker;

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by theformula:

A-B-DE-F)_(q)

wherein

A is the modified oligonucleotide;

B is the cleavable moiety;

D is the branching group;

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by theformula:

A-BE-F)_(q)

wherein

A is the modified oligonucleotide;

B is the cleavable moiety;

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, the compound has a structure represented by theformula:

A-DE-F)_(q)

wherein

A is the modified oligonucleotide;

D is the branching group;

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, the conjugate linker has a structure selectedfrom among:

wherein each L is, independently, a phosphorus linking group or aneutral linking group; and

each n is, independently, from 1 to 20.

In certain embodiments, the conjugate linker has a structure selectedfrom among:

In certain embodiments, the conjugate linker has the followingstructure:

In certain embodiments, the conjugate linker has a structure selectedfrom among:

In certain embodiments, the conjugate linker has a structure selectedfrom among:

In certain embodiments, the conjugate linker has a structure selectedfrom among:

In certain embodiments, the conjugate linker comprises a pyrrolidine. Incertain embodiments, the conjugate linker does not comprise apyrrolidine.In certain embodiments, the conjugate linker comprises PEG.In certain embodiments, the conjugate linker comprises an amide. Incertain embodiments, the conjugate linker comprises at least two amides.In certain embodiments, the conjugate linker does not comprise an amide.In certain embodiments, the conjugate linker comprises a polyamide.In certain embodiments, the conjugate linker comprises an amine.

In certain embodiments, the conjugate linker comprises one or moredisulfide bonds.

In certain embodiments, the conjugate linker comprises a protein bindingmoiety. In certain embodiments, the protein binding moiety comprises alipid. In certain embodiments, the protein binding moiety is selectedfrom among: cholesterol, cholic acid, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol,geranyloxyhexyl group, hexadecylglycerol, borneol, menthol,1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl,or phenoxazine), a vitamin (e.g., folate, vitamin A, vitamin E, biotin,pyridoxal), a peptide, a carbohydrate (e.g., monosaccharide,disaccharide, trisaccharide, tetrasaccharide, oligosaccharide,polysaccharide), an endosomolytic component, a steroid (e.g., uvaol,hecigenin, diosgenin), a terpene (e.g., triterpene, e.g.,sarsasapogenin, friedelin, epifriedelanol derivatized lithocholic acid),or a cationic lipid. In certain embodiments, the protein binding moietyis selected from among: a C16 to C22 long chain saturated or unsaturatedfatty acid, cholesterol, cholic acid, vitamin E, adamantane or1-pentafluoropropyl.

In certain embodiments, the conjugate linker has a structure selectedfrom among:

wherein each n is, independently, is from 1 to 20; and p is from 1 to 6.

In certain embodiments, the conjugate linker has a structure selectedfrom among:

wherein each n is, independently, from 1 to 20.

In certain embodiments, the conjugate linker has a structure selectedfrom among:

In certain embodiments, the conjugate linker has a structure selectedfrom among:

wherein n is from 1 to 20.

In certain embodiments, the conjugate linker has a structure selectedfrom among:

In certain embodiments, the conjugate linker has a structure selectedfrom among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

In certain embodiments, the conjugate linker has the followingstructure:

In certain embodiments, the branching group has one of the followingstructures:

wherein each A₁ is independently, O, S, C═O or NH; and

each n is, independently, from 1 to 20.

In certain embodiments, the branching group has one of the followingstructures:

wherein each A₁ is independently, O, S, C═O or NH; and

each n is, independently, from 1 to 20.

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group comprises an ether.In certain embodiments, the branching group has the following structure:

each n is, independently, from 1 to 20; and

m is from 2 to 6.

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group has the following structure:

In certain embodiments, the branching group comprises:

wherein each j is an integer from 1 to 3; andwherein each n is an integer from 1 to 20.In certain embodiments, the branching group comprises:

In certain embodiments, each tether is selected from among:

wherein

-   -   L is selected from a phosphorus linking group and a neutral        linking group;    -   Z₁ is C(═O)O—R₂;    -   Z₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky;    -   R₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky; and    -   each m₁ is, independently, from 0 to 20 wherein at least one m₁        is greater than 0 for each tether.        In certain embodiments, each tether is selected from among:

wherein Z₂ is H or CH₃; and

each m₂ is, independently, from 0 to 20 wherein at least one m₂ isgreater than 0 for each tether.

In certain embodiments, each tether is selected from among:

wherein n is from 1 to 12; and

wherein m is from 1 to 12.

In certain embodiments, at least one tether comprises ethylene glycol.In certain embodiments, at least one tether comprises an amide. Incertain embodiments, at least one tether comprises a polyamide.In certain embodiments, at least one tether comprises an amine.In certain embodiments, at least two tethers are different from oneanother. In certain embodiments, all of the tethers are the same as oneanother.In certain embodiments, each tether is selected from among:

wherein each n is, independently, from 1 to 20; and

each p is from 1 to about 6.

In certain embodiments, each tether is selected from among:

In certain embodiments, each tether has the following structure:

wherein each n is, independently, from 1 to 20.

In certain embodiments, each tether has the following structure:

In certain embodiments, the tether has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.In certain embodiments, the tether has a structure selected from among:

In certain embodiments, the ligand is galactose.In certain embodiments, the ligand is mannose-6-phosphate.In certain embodiments, each ligand is selected from among:

wherein each R₁ is selected from OH and NHCOOH.

In certain embodiments, each ligand is selected from among:

In certain embodiments, each ligand has the following structure:

In certain embodiments, each ligand has the following structure:

In certain embodiments, the conjugate group comprises a cell-targetingmoiety.In certain embodiments, the conjugate group comprises a cell-targetingmoiety having the following structure:

wherein each n is, independently, from 1 to 20.

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety has the followingstructure:

wherein each n is, independently, from 1 to 20.

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety comprises:

In certain embodiments, the cell-targeting moiety has the followingstructure:

In certain embodiments, the cell-targeting moiety comprises:

wherein each Y is selected from O, S, a substituted or unsubstitutedC₁-C₁₀ alkyl, amino, substituted amino, azido, alkenyl or alkynyl.In certain embodiments, the conjugate group comprises:

wherein each Y is selected from O, S, a substituted or unsubstitutedC₁-C₁₀ alkyl, amino, substituted amino, azido, alkenyl or alkynyl.In certain embodiments, the cell-targeting moiety has the followingstructure:

wherein each Y is selected from O, S, a substituted or unsubstitutedC₁-C₁₀ alkyl, amino, substituted amino, azido, alkenyl or alkynyl.In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

T In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises:

In certain embodiments, the conjugate group comprises a cleavable moietyselected from among: a phosphodiester, an amide, or an ester.In certain embodiments, the conjugate group comprises a phosphodiestercleavable moiety.In certain embodiments, the conjugate group does not comprise acleavable moiety, and wherein the conjugate group comprises aphosphorothioate linkage between the conjugate group and theoligonucleotide.In certain embodiments, the conjugate group comprises an amide cleavablemoiety.In certain embodiments, the conjugate group comprises an ester cleavablemoiety.In certain embodiments, the compound has the following structure:

wherein each n is, independently, from 1 to 20;

Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein each n is, independently, from 1 to 20;

Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein each n is, independently, from 1 to 20;

Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide;

Z is H or a linked solid support; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein each n is, independently, from 1 to 20;

Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide;

Z is H or a linked solid support; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the conjugate group comprises:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the conjugate group comprises:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, the conjugate group comprises:

wherein Q₁₃ is H or O(CH₂)₂—OCH₃;

A is the modified oligonucleotide; and

Bx is a heterocyclic base moiety.

In certain embodiments, B_(x) is selected from among from adenine,guanine, thymine, uracil, or cytosine, or 5-methyl cytosine. In certainembodiments, B_(x) is adenine. In certain embodiments, B_(x) is thymine.In certain embodiments, Q₁₃ is O(CH₂)₂—OCH₃. In certain embodiments, Q₁₃is H.

Certain embodiments of the invention provide a prodrug comprising thecompositions or compounds disclosed herein.

In certain embodiments, the compound is in a salt form. In furtherembodiments, the compound further comprises of a pharmaceuticallyacceptable carrier or diluent. In certain embodiments, the compoundcomprises a modified oligonucleotide targeting ApoCIII and a conjugategroup, or a salt thereof, and a pharmaceutically acceptable carrier ordiluent.

Certain embodiments provide compositions and methods comprisingadministering to an animal a conjugated antisense compound orcomposition disclosed herein. In certain embodiments, administering theconjugated antisense compound prevents, treats, ameliorates, or slowsprogression of a cardiovascular, metabolic and/or inflammatory disease.

Certain embodiments provide compositions and methods for use in therapyto treat an ApoCIII related disease, disorder or condition. In certainembodiments, the ApoCIII levels are elevated in an animal. In certainembodiments, the composition is a compound comprising an ApoCIIIspecific inhibitor. In certain embodiments, the ApoCIII specificinhibitor is a nucleic acid. In certain embodiments, the nucleic acid isan antisense compound. In certain embodiments, the antisense compound isa modified oligonucleotide targeting ApoCIII. In certain embodiments,the antisense compound is a modified oligonucleotide targeting ApoCIIIand a conjugate group. In certain embodiments, the modifiedoligonucleotide targeting ApoCIII with the conjugate group, is used intreating, preventing, slowing progression, ameliorating an inflammatory,cardiovascular and/or metabolic disease, disorder or condition. Incertain embodiments, the compositions and methods for therapy includeadministering an ApoCIII specific inhibitor to an individual in needthereof.

Certain embodiments provide conjugated antisense compounds andcompositions and methods for reducing ApoCIII levels. In certainembodiments, ApoCIII levels are reduced in the liver, adipose tissue,heart, skeletal muscle or small intestine.

In certain embodiments, reducing ApoCIII levels in a tissue, organ orsubject increases HDL levels. In certain embodiments, the HDL levels areincreased by at least 90%, by at least 80%, by at least 70%, by at least60%, by at least 50%, by at least 45%, at least 40%, by at least 35%, byat least 30%, by at least 25%, by at least 20%, by at least 15%, by atleast 10% or by at least 5% from the baseline HDL level.

In certain embodiments, reducing ApoCIII levels in a tissue, organ orsubject reduces TG levels. In certain embodiments, the subject has atriglyceride level ≥100 mg/dL, ≥200 mg/dL, ≥300 mg/dL, ≥400 mg/dL, ≥440mg/dL, ≥500 mg/dL, ≥600 mg/dL, ≥700 mg/dL, ≥800 mg/dL, ≥880 mg/dL, ≥900mg/dL, ≥1000 mg/dL, ≥1100 mg/dL, ≥1200 mg/dL, ≥1300 mg/dL, ≥1400 mg/dL,≥1500 mg/dL, ≥1600 mg/dL, ≥1700 mg/dL, ≥1800 mg/dL, ≥1900 mg/dL, ≥2000mg/dL.

In certain embodiments, the TG levels (postprandial or fasting) aredecreased by at least 90%, by at least 80%, by at least 70%, by at least60%, by at least 50%, by at least 45%, at least 40%, by at least 35%, byat least 30%, by at least 25%, by at least 20%, by at least 15%, by atleast 10%, by at least 5% or by at least 1% from the baseline TG level.In certain embodiments, the TG (postprandial or fasting) level isdecreased to ≤1900 mg/dL, ≤1800 mg/dL, ≤1700 mg/dL, ≤1600 mg/dL, ≤1500mg/dL, ≤1400 mg/dL, ≤1300 mg/dL, ≤1200 mg/dL, ≤1100 mg/dL, ≤1000 mg/dL,≤900 mg/dL, ≤800 mg/dL, ≤750 mg/dL, ≤700 mg/dL, ≤650 mg/dL, ≤600 mg/dL,≤550 mg/dL, ≤500 mg/dL, ≤450 mg/dL, ≤400 mg/dL, ≤350 mg/dL, ≤300 mg/dL,≤250 mg/dL, ≤200 mg/dL, ≤150 mg/dL or ≤100 mg/dL.

In certain embodiments, reducing ApoCIII levels in a tissue, organ orsubject improves the ratio of

LDL to HDL or the ratio of TG to HDL.

In certain embodiments, reducing ApoCIII levels in a tissue, organ orsubject improves insulin sensitivity.

In certain embodiments, reducing ApoCIII levels in a tissue, organ orsubject increases chylomicron clearance.

Certain embodiments provide compositions and methods to reduce ApoCIIImRNA or protein expression in an animal comprising administering to theanimal a conjugated antisense compound or composition disclosed hereinto reduce ApoCIII mRNA or protein expression in the animal.

Certain embodiments provide conjugated antisense compounds andcompositions and methods for preventing, treating, delaying, slowing theprogression and/or ameliorating ApoCIII related diseases, disorders, andconditions in a subject in need thereof. In certain embodiments, suchdiseases, disorders, and conditions include inflammatory, cardiovascularand/or metabolic diseases, disorders, and conditions. Certain suchcardiovascular diseases, disorders or conditions include, but are notlimited to, chylomicronemia, hypertriglyceridemia, aortic stenosis,aneurysm (e.g., abdominal aortic aneurysm), angina, arrhythmia,atherosclerosis, cerebrovascular disease, coronary artery disease,coronary heart disease, dyslipidemia, hypercholesterolemia,hyperlipidemia, hypertension, myocardial infarction, peripheral vasculardisease (e.g., peripheral artery disease, peripheral artery occlusivedisease), Fredrickson Type I dyslipidemia, FCS, LPL deficiency, retinalvascular occlusion, or stroke. Certain such metabolic diseases,disorders or conditions include, but are not limited to, hyperglycemia,prediabetes, diabetes (type I and type II), obesity, insulin resistance,metabolic syndrome and diabetic dyslipidemia. Certain such inflammatorydiseases, disorders or conditions include, but are not limited to,pancreatitis, aortic stenosis, coronary artery disease (CAD),Alzheimer's Disease and thromboembolic diseases, disorder or conditions.Certain thromboembolic diseases, disorders or conditions include, butare not limited to, stroke, thrombosis (e.g., venous thromboembolism),myocardial infarction and peripheral vascular disease. Certainembodiments provide conjugated antisense compounds and compositions andmethods for preventing, treating, delaying, slowing the progressionand/or ameliorating hypertriglyceridemia. Certain embodiments provideconjugated antisense compounds and compositions and methods forpreventing, treating, delaying, slowing the progression and/orameliorating chylomicronemia. Certain embodiments provide conjugatedantisense compounds and compositions and methods for preventing,treating, delaying, slowing the progression and/or amelioratingpancreatitis.

Certain embodiments provide a method of reducing at least one symptom ofa cardiovascular disease, disorder or condition. In certain embodiments,the symptoms include, but are not limited to, angina, chest pain,shortness of breath, palpitations, weakness, dizziness, nausea,sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation,swelling in the lower extremities, cyanosis, fatigue, fainting, numbnessof the face, numbness of the limbs, claudication or cramping of muscles,bloating of the abdomen, and fever. In certain embodiments, symptoms ofa metabolic disease, disorder or condition include, but are not limitedto, frequent urination, unusual thirst, extreme hunger, unusual weightloss, extreme fatigue, irritability, frequent infections, blurredvision, cuts/bruises that are slow to heal, tingling/numbness in thehands/feet and recurring skin, gum, or bladder infections. Certainembodiments provide a method of reducing at least one symptom ofhypertriglyceridemia. Certain embodiments provide a method of reducingat least one symptom of chylomicronemia. Certain embodiments provide amethod of reducing at least one symptom of pancreatitis.

In certain embodiments, the modulation of ApoCIII expression occurs in acell, tissue or organ. In certain embodiments, the modulations occur ina cell, tissue or organ in an animal. In certain embodiments, themodulation is a reduction in ApoCIII mRNA level. In certain embodiments,the modulation is a reduction in ApoCIII protein level. In certainembodiments, both ApoCIII mRNA and protein levels are reduced. Suchreduction may occur in a time-dependent or in a dose-dependent manner.

In certain embodiments, the subject or animal is human.

In certain embodiments, the compound is parenterally administered. Infurther embodiments, the parenteral administration is subcutaneous.

In certain embodiments, the conjugated antisense compound or compositionis co-administered with a second agent or therapy. In certainembodiments, the conjugated antisense compound or composition and thesecond agent are administered concomitantly.

In certain embodiments, the second agent is a glucose-lowering agent. Incertain embodiments, the second agent is a LDL, TG or cholesterollowering agent. In certain embodiments, the second agent is ananti-inflammatory agent. In certain embodiments, the second agent is anAlzheimer Disease drug. In certain embodiments, the second agent can be,but is not limited to, a non-steroidal anti-inflammatory drug (NSAIDe.g., aspirin), niacin (e.g., Niaspan), nicotinic acid, an apoBinhibitor (e.g., Mipomersen), a CETP inhibitor (e.g., Anacetrapib), anapo(a) inhibitor, a thyroid hormone analog (e.g., Eprotirome), a HMG-CoAreductase inhibitor (e.g., a statin), a fibrate (e.g., Gemfibrozil) andan microsomal triglyceride transfer protein inhibitor (e.g.,Lomitapide). Agents or therapies can be co-administered or administeredconcomitantly. Agents or therapies can be sequentially or subsequentlyadministered.

Certain embodiments provide use of the compositions and conjugatedantisense compounds described herein targeted to ApoCIII for decreasingApoCIII levels in an animal. Certain embodiments provide use of acompound targeted to ApoCIII for decreasing ApoCIII levels in an animal.Certain embodiments provide use of a compound targeted to ApoCIII forincreasing HDL levels in an animal. Certain embodiments provide use of acompound targeted to ApoCIII for increasing HDL chylomicron clearance inan animal. Certain embodiments provide use of a compounds targeted toApoCIII for the treatment, prevention, or amelioration of a disease,disorder, or condition associated with ApoCIII. Certain embodimentsprovide use of a compound targeted to ApoCIII for the treatment,prevention, or amelioration of a hypertriglyceridemia. Certainembodiments provide use of a compound targeted to ApoCIII for thetreatment, prevention, or amelioration of a chylomicronemia (e.g., FCSand/or LPLD). Certain embodiments provide use of a compound targeted toApoCIII for the treatment, prevention, or amelioration of apancreatitis.

Certain embodiments provide use of the compositions and conjugatedantisense compounds described herein targeted to ApoCIII in thepreparation of a medicament for decreasing ApoCIII levels in an animal.Certain embodiments provide use of the compositions and compounds forthe preparation of a medicament for the treatment, prevention, oramelioration of a disease, disorder, or condition associated withApoCIII.

Certain embodiments provide the use of the compositions and conjugatedantisense compounds as described herein in the manufacture of amedicament for treating, ameliorating, delaying or preventing one ormore of a disease related to ApoCIII.

Certain embodiments provide a kit for treating, preventing, orameliorating a disease, disorder or condition as described hereinwherein the kit comprises: (i) an ApoCIII specific inhibitor asdescribed herein; and optionally (ii) a second agent or therapy asdescribed herein.

A kit of the present invention can further include instructions forusing the kit to treat, prevent, or ameliorate a disease, disorder orcondition as described herein by combination therapy as describedherein.

B. Certain Compounds

In certain embodiments, the invention provides conjugated antisensecompounds comprising antisense oligonucleotides and a conjugate.

a. Certain Antisense Oligonucleotides

In certain embodiments, the invention provides antisenseoligonucleotides. Such antisense oligonucleotides comprise linkednucleosides, each nucleoside comprising a sugar moiety and a nucleobase.The structure of such antisense oligonucleotides may be considered interms of chemical features (e.g., modifications and patterns ofmodifications) and nucleobase sequence (e.g., sequence of antisenseoligonucleotide, identity and sequence of target nucleic acid).

i. Certain Chemistry Features

In certain embodiments, antisense oligonucleotide comprise one or moremodification. In certain such embodiments, antisense oligonucleotidescomprise one or more modified nucleosides and/or modifiedinternucleoside linkages. In certain embodiments, modified nucleosidescomprise a modified sugar moirty and/or modified nucleobase.

1. Certain Sugar Moieties

In certain embodiments, compounds of the disclosure comprise one or moremodified nucleosides comprising a modified sugar moiety. Such compoundscomprising one or more sugar-modified nucleosides may have desirableproperties, such as enhanced nuclease stability or increased bindingaffinity with a target nucleic acid relative to an oligonucleotidecomprising only nucleosides comprising naturally occurring sugarmoieties. In certain embodiments, modified sugar moieties aresubstituted sugar moieties. In certain embodiments, modified sugarmoieties are sugar surrogates. Such sugar surrogates may comprise one ormore substitutions corresponding to those of substituted sugar moieties.

In certain embodiments, modified sugar moieties are substituted sugarmoieties comprising one or more non-bridging sugar substituent,including but not limited to substituents at the 2′ and/or 5′ positions.Examples of sugar substituents suitable for the 2′-position, include,but are not limited to: 2′-F, 2′-OCH₃ (“OMe” or “O-methyl”), and2′-O(CH₂)₂OCH₃ (“MOE”). In certain embodiments, sugar substituents atthe 2′ position is selected from allyl, amino, azido, thio, O-allyl,O—C₁-C₁₀ alkyl, O—C₁-C₁₀ substituted alkyl; OCF₃, O(CH₂)₂SCH₃,O(CH₂)₂—O—N(Rm)(Rn), and O—CH₂—C(═O)—N(Rm)(Rn), where each Rm and Rn is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl. Examplesof sugar substituents at the 5′-position, include, but are not limitedto: 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy. In certainembodiments, substituted sugars comprise more than one non-bridgingsugar substituent, for example, 2′-F-5′-methyl sugar moieties (see,e.g., PCT International Application WO 2008/101157, for additional 5′,2′-bis substituted sugar moieties and nucleosides).

Nucleosides comprising 2′-substituted sugar moieties are referred to as2′-substituted nucleosides. In certain embodiments, a 2′-substitutednucleoside comprises a 2′-substituent group selected from halo, allyl,amino, azido, SH, CN, OCN, CF₃, OCF₃, O, S, or N(R_(m))-alkyl; O, S, orN(R_(m))-alkenyl; O, S or N(R_(m))-alkynyl; O-alkylenyl-O-alkyl,alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R_(n) is, independently, H, an amino protecting group orsubstituted or unsubstituted C₁-C₁₀ alkyl. These 2′-substituent groupscan be further substituted with one or more substituent groupsindependently selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro (NO₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl,alkenyl and alkynyl.

In certain embodiments, a 2′-substituted nucleoside comprises a2′-substituent group selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂,CH₂—CH═CH₂, O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, OCF₃, O—CH₃,OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N(CH₃)₂,and O—CH₂—C(═O)—N(H)CH₃.

In certain embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, O—CH₃, andOCH₂CH₂OCH₃.

Certain modified sugar moieties comprise a bridging sugar substituentthat forms a second ring resulting in a bicyclic sugar moiety. Incertain such embodiments, the bicyclic sugar moiety comprises a bridgebetween the 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′sugar substituents, include, but are not limited to:—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a)R_(b))]_(n)—O—, —C(R_(a)R_(b))—N(R)—O—or, —C(R_(a)R_(b))—O—N(R)—; 4′-CH₂- 2′, 4′-(CH₂)₂-2′,4′—(CH₂)₃-2′,4′—(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2; 4′—(CH₂)₂—O-2′ (ENA);4′-CH(CH₃)—O-2′ (cEt) and 4′-CH(CH₂OCH₃)—O-2′, and analogs thereof (see,e.g., U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008);4′-C(CH₃)(CH₃)—O-2′ and analogs thereof, (see, e.g., WO2009/006478,published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ and analogs thereof (see,e.g., WO2008/150729, published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (see,e.g., US2004/0171570, published Sep. 2, 2004); 4′-CH₂—O—N(R)-2′, and4′-CH₂—N(R)—O-2′-, wherein each R is, independently, H, a protectinggroup, or C₁-C₁₂ alkyl; 4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂ alkyl,or a protecting group (see, U.S. Pat. No. 7,427,672, issued on Sep. 23,2008); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Chattopadhyaya, et al., J. Org.Chem., 2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ and analogs thereof(see, published PCT International Application WO 2008/154401, publishedon Dec. 8, 2008).

In certain embodiments, such 4′ to 2′ bridges independently comprisefrom 1 to 4 linked groups independently selected from—[C(R_(a))(R_(b))]_(n)—, —C(R_(a))═C(R_(b))—, —C(R_(a))═N—,—C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—, —S(═O)_(x)—, and—N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₁₂)aryl, substituted C₅-C₂₀ aryl, heterocycle radical, substitutedheterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclicradical, substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁,N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁),or sulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl,or a protecting group.

Nucleosides comprising bicyclic sugar moieties are referred to asbicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are notlimited to, (A) α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-Methyleneoxy(4′-CH₂—O-2′) BNA (also referred to as locked nucleic acid or LNA), (C)Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) Aminooxy (4′-CH₂—O—N(R)-2′) BNA,(E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F) Methyl(methyleneoxy)(4′-CH(CH₃)—O-2′) BNA (also referred to as constrained ethyl or cEt),(G) methylene-thio (4′-CH₂—S-2′) BNA, (H) methylene-amino(4′-CH₂—N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA,and (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA as depicted below.

wherein Bx is a nucleobase moiety and R is, independently, H, aprotecting group, or C₁-C₁₂ alkyl.

Additional bicyclic sugar moieties are known in the art, for example:Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al.,Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad.Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem.Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63,10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379(Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2,558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr.Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 7,053,207,6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and7,399,845; WO 2004/106356, WO 1994/14226, WO 2005/021570, and WO2007/134181; U.S. Patent Publication Nos. US2004/0171570,US2007/0287831, and US2008/0039618; U.S. patent Ser. Nos. 12/129,154,60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787,and 61/099,844; and PCT International Applications Nos.PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922.

In certain embodiments, bicyclic sugar moieties and nucleosidesincorporating such bicyclic sugar moieties are further defined byisomeric configuration. For example, a nucleoside comprising a 4′-2′methylene-oxy bridge, may be in the α-L configuration or in the β-Dconfiguration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) bicyclicnucleosides have been incorporated into antisense oligonucleotides thatshowed antisense activity (Frieden et al., Nucleic Acids Research, 2003,21, 6365-6372).

In certain embodiments, substituted sugar moieties comprise one or morenon-bridging sugar substituent and one or more bridging sugarsubstituent (e.g., 5′-substituted and 4′-2′ bridged sugars). (see, PCTInternational Application WO 2007/134181, published on Nov. 22, 2007,wherein LNA is substituted with, for example, a 5′-methyl or a 5′-vinylgroup).

In certain embodiments, modified sugar moieties are sugar surrogates. Incertain such embodiments, the oxygen atom of the naturally occurringsugar is substituted, e.g., with a sulfur, carbon or nitrogen atom. Incertain such embodiments, such modified sugar moiety also comprisesbridging and/or non-bridging substituents as described above. Forexample, certain sugar surrogates comprise a 4′-sulfur atom and asubstitution at the 2′-position (see, e.g., published U.S. PatentApplication US2005/0130923, published on Jun. 16, 2005) and/or the 5′position. By way of additional example, carbocyclic bicyclic nucleosideshaving a 4′-2′ bridge have been described (see, e.g., Freier et al.,Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J.Org. Chem., 2006, 71, 7731-7740).

In certain embodiments, sugar surrogates comprise rings having otherthan 5-atoms. For example, in certain embodiments, a sugar surrogatecomprises a morpholino. Morpholino compounds and their use in oligomericcompounds has been reported in numerous patents and published articles(see for example: Braasch et al., Biochemistry, 2002, 41, 4503-4510; andU.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506). As usedhere, the term “morpholino” means a sugar surrogate having the followingstructure:

In certain embodiments, morpholinos may be modified, for example byadding or altering various substituent groups from the above morpholinostructure. Such sugar surrogates are referred to herein as “modifiedmorpholinos.”

For another example, in certain embodiments, a sugar surrogate comprisesa six-membered tetrahydropyran. Such tetrahydropyrans may be furthermodified or substituted. Nucleosides comprising such modifiedtetrahydropyrans include, but are not limited to, hexitol nucleic acid(HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (seeLeumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA(F-HNA), and those compounds having Formula VI:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula VI:

Bx is a nucleobase moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the antisense compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the antisense compound and theother of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup, or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each, independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl, or substituted C₂-C₆ alkynyl; and each of R₁ and R₂ isindependently selected from among: hydrogen, halogen, substituted orunsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂,NJ₃C(═X)NJ₁J₂, and CN, wherein X is O, S or NJ₁, and each J₁, J₂, and J₃is, independently, H or C₁-C₆ alkyl.

In certain embodiments, the modified THP nucleosides of Formula VI areprovided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other thanH. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇is methyl. In certain embodiments, THP nucleosides of Formula VI areprovided wherein one of R₁ and R₂ is F. In certain embodiments, R₁ isfluoro and R₂ is H, R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxyand R₂ is H.

Many other bicyclo and tricyclo sugar surrogate ring systems are alsoknown in the art that can be used to modify nucleosides forincorporation into antisense compounds (see, e.g., review article:Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 Published on Aug. 21, 2008 for otherdisclosed 5′, 2′-bis substituted nucleosides) and replacement of theribosyl ring oxygen atom with S and further substitution at the2′-position (see published U.S. Patent Application US2005-0130923,published on Jun. 16, 2005) or alternatively 5′-substitution of abicyclic nucleic acid (see PCT International Application WO 2007/134181,published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside isfurther substituted at the 5′ position with a 5′-methyl or a 5′-vinylgroup). The synthesis and preparation of carbocyclic bicyclicnucleosides along with their oligomerization and biochemical studieshave also been described (see, e.g., Srivastava et al., J. Am. Chem.Soc. 2007, 129(26), 8362-8379).

In certain embodiments, the present disclosure provides oligonucleotidescomprising modified nucleosides. Those modified nucleotides may includemodified sugars, modified nucleobases, and/or modified linkages. Thespecific modifications are selected such that the resultingoligonucleotides possess desirable characteristics. In certainembodiments, oligonucleotides comprise one or more RNA-like nucleosides.In certain embodiments, oligonucleotides comprise one or more DNA-likenucleotides.

2. Certain Nucleobase Modifications

In certain embodiments, nucleosides of the present disclosure compriseone or more unmodified nucleobases. In certain embodiments, nucleosidesof the present disclosure comprise one or more modified nucleobases.

In certain embodiments, modified nucleobases are selected from:universal bases, hydrophobic bases, promiscuous bases, size-expandedbases, and fluorinated bases as defined herein. 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine;5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases,hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases as defined herein. Further modified nucleobasesinclude tricyclic pyrimidines such as phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′-4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz,J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613; and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, Crooke, S. T. and Lebleu, B., Eds., CRCPress, 1993, 273-288.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include without limitation, U.S. Pat. Nos.3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985;5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference in its entirety.

3. Certain Internucleoside Linkages

In certain embodiments, the present disclosure provides oligonucleotidescomprising linked nucleosides. In such embodiments, nucleosides may belinked together using any internucleoside linkage. The two main classesof internucleoside linking groups are defined by the presence or absenceof a phosphorus atom. Representative phosphorus containinginternucleoside linkages include, but are not limited to,phosphodiesters (PO), phosphotriesters, methylphosphonates,phosphoramidate, and phosphorothioates (PS). Representativenon-phosphorus containing internucleoside linking groups include, butare not limited to, methylenemethylimino (—CH₂—N(CH₃)—O—CH₂—),thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane(—O—Si(H)₂—O—); and N,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—).Modified linkages, compared to natural phosphodiester linkages, can beused to alter, typically increase, nuclease resistance of theoligonucleotide. In certain embodiments, internucleoside linkages havinga chiral atom can be prepared as a racemic mixture, or as separateenantiomers. Representative chiral linkages include, but are not limitedto, alkylphosphonates and phosphorothioates. Methods of preparation ofphosphorous-containing and non-phosphorous-containing internucleosidelinkages are well known to those skilled in the art.

The oligonucleotides described herein contain one or more asymmetriccenters and thus give rise to enantiomers, diastereomers, and otherstereoisomeric configurations that may be defined, in terms of absolutestereochemistry, as (R) or (S), α or β such as for sugar anomers, or as(D) or (L) such as for amino acids etc. Included in the antisensecompounds provided herein are all such possible isomers, as well astheir racemic and optically pure forms.

Neutral internucleoside linkages include without limitation,phosphotriesters, methylphosphonates, MMI (3′-CH₂—N(CH₃)—O-5′), amide-3(3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal(3′-O—CH₂—O-5′), and thioformacetal (3′-S—CH₂—O-5′). Further neutralinternucleoside linkages include nonionic linkages comprising siloxane(dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonateester and amides (See for example: Carbohydrate Modifications inAntisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS SymposiumSeries 580; Chapters 3 and 4, 40-65). Further neutral internucleosidelinkages include nonionic linkages comprising mixed N, O, S and CH₂component parts.

4. Certain Motifs

In certain embodiments, antisense oligonucleotides comprise one or moremodified nucleoside (e.g., nucleoside comprising a modified sugar and/ormodified nucleobase) and/or one or more modified internucleosidelinkage. The pattern of such modifications on an oligonucleotide isreferred to herein as a motif. In certain embodiments, sugar,nucleobase, and linkage motifs are independent of one another.

a. Certain Sugar Motifs

In certain embodiments, oligonucleotides comprise one or more type ofmodified sugar moieties and/or naturally occurring sugar moietiesarranged along an oligonucleotide or region thereof in a defined patternor sugar modification motif. Such motifs may include any of the sugarmodifications discussed herein and/or other known sugar modifications.

In certain embodiments, the oligonucleotides comprise or consist of aregion having a gapmer sugar motif, which comprises two external regionsor “wings” and a central or internal region or “gap.” The three regionsof a gapmer sugar motif (the 5′-wing, the gap, and the 3′-wing) form acontiguous sequence of nucleosides wherein at least some of the sugarmoieties of the nucleosides of each of the wings differ from at leastsome of the sugar moieties of the nucleosides of the gap. Specifically,at least the sugar moieties of the nucleosides of each wing that areclosest to the gap (the 3′-most nucleoside of the 5′-wing and the5′-most nucleoside of the 3′-wing) differ from the sugar moiety of theneighboring gap nucleosides, thus defining the boundary between thewings and the gap. In certain embodiments, the sugar moieties within thegap are the same as one another. In certain embodiments, the gapincludes one or more nucleoside having a sugar moiety that differs fromthe sugar moiety of one or more other nucleosides of the gap. In certainembodiments, the sugar motifs of the two wings are the same as oneanother (symmetric sugar gapmer). In certain embodiments, the sugarmotifs of the 5′-wing differs from the sugar motif of the 3′-wing(asymmetric sugar gapmer).

i. Certain 5′-Wings

In certain embodiments, the 5′-wing of a gapmer consists of 1 to 8linked nucleosides. In certain embodiments, the 5′-wing of a gapmerconsists of 1 to 7 linked nucleosides. In certain embodiments, the5′-wing of a gapmer consists of 1 to 6 linked nucleosides. In certainembodiments, the 5′-wing of a gapmer consists of 1 to 5 linkednucleosides. In certain embodiments, the 5′-wing of a gapmer consists of2 to 5 linked nucleosides. In certain embodiments, the 5′-wing of agapmer consists of 3 to 5 linked nucleosides. In certain embodiments,the 5′-wing of a gapmer consists of 4 or 5 linked nucleosides. Incertain embodiments, the 5′-wing of a gapmer consists of 1 to 4 linkednucleosides. In certain embodiments, the 5′-wing of a gapmer consists of1 to 3 linked nucleosides. In certain embodiments, the 5′-wing of agapmer consists of 1 or 2 linked nucleosides. In certain embodiments,the 5′-wing of a gapmer consists of 2 to 4 linked nucleosides. Incertain embodiments, the 5′-wing of a gapmer consists of 2 or 3 linkednucleosides. In certain embodiments, the 5′-wing of a gapmer consists of3 or 4 linked nucleosides. In certain embodiments, the 5′-wing of agapmer consists of 1 nucleoside. In certain embodiments, the 5′-wing ofa gapmer consists of 2 linked nucleosides. In certain embodiments, the5′-wing of a gapmer consists of 3 linked nucleosides. In certainembodiments, the 5′-wing of a gapmer consists of 4 linked nucleosides.In certain embodiments, the 5′-wing of a gapmer consists of 5 linkednucleosides. In certain embodiments, the 5′-wing of a gapmer consists of6 linked nucleosides.

In certain embodiments, the 5′-wing of a gapmer comprises at least onebicyclic nucleoside. In certain embodiments, the 5′-wing of a gapmercomprises at least two bicyclic nucleosides. In certain embodiments, the5′-wing of a gapmer comprises at least three bicyclic nucleosides. Incertain embodiments, the 5′-wing of a gapmer comprises at least fourbicyclic nucleosides. In certain embodiments, the 5′-wing of a gapmercomprises at least one constrained ethyl nucleoside. In certainembodiments, the 5′-wing of a gapmer comprises at least one LNAnucleoside. In certain embodiments, each nucleoside of the 5′-wing of agapmer is a bicyclic nucleoside. In certain embodiments, each nucleosideof the 5′-wing of a gapmer is a constrained ethyl nucleoside. In certainembodiments, each nucleoside of the 5′-wing of a gapmer is a LNAnucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least onenon-bicyclic modified nucleoside. In certain embodiments, the 5′-wing ofa gapmer comprises at least one 2′-substituted nucleoside. In certainembodiments, the 5′-wing of a gapmer comprises at least one 2′-MOEnucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one 2′-OMe nucleoside. In certain embodiments, each nucleoside ofthe 5′-wing of a gapmer is a non-bicyclic modified nucleoside. Incertain embodiments, each nucleoside of the 5′-wing of a gapmer is a2′-substituted nucleoside. In certain embodiments, each nucleoside ofthe 5′-wing of a gapmer is a 2′-MOE nucleoside. In certain embodiments,each nucleoside of the 5′-wing of a gapmer is a 2′-OMe nucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least one2′-deoxynucleoside. In certain embodiments, each nucleoside of the5′-wing of a gapmer is a 2′-deoxynucleoside. In a certain embodiments,the 5′-wing of a gapmer comprises at least one ribonucleoside. Incertain embodiments, each nucleoside of the 5′-wing of a gapmer is aribonucleoside. In certain embodiments, one, more than one, or each ofthe nucleosides of the 5′-wing is an RNA-like nucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one non-bicyclic modified nucleoside.In certain embodiments, the 5′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one 2′-substituted nucleoside. Incertain embodiments, the 5′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one 2′-MOE nucleoside. In certainembodiments, the 5′-wing of a gapmer comprises at least one bicyclicnucleoside and at least one 2′-OMe nucleoside. In certain embodiments,the 5′-wing of a gapmer comprises at least one bicyclic nucleoside andat least one 2′-deoxynucleoside.

In certain embodiments, the 5′-wing of a gapmer comprises at least oneconstrained ethyl nucleoside and at least one non-bicyclic modifiednucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-substitutednucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-MOEnucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-OMenucleoside. In certain embodiments, the 5′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one2′-deoxynucleoside.

ii. Certain 3′-Wings

In certain embodiments, the 3′-wing of a gapmer consists of 1 to 8linked nucleosides. In certain embodiments, the 3′-wing of a gapmerconsists of 1 to 7 linked nucleosides. In certain embodiments, the3′-wing of a gapmer consists of 1 to 6 linked nucleosides. In certainembodiments, the 3′-wing of a gapmer consists of 1 to 5 linkednucleosides. In certain embodiments, the 3′-wing of a gapmer consists of2 to 5 linked nucleosides. In certain embodiments, the 3′-wing of agapmer consists of 3 to 5 linked nucleosides. In certain embodiments,the 3′-wing of a gapmer consists of 4 or 5 linked nucleosides. Incertain embodiments, the 3′-wing of a gapmer consists of 1 to 4 linkednucleosides. In certain embodiments, the 3′-wing of a gapmer consists of1 to 3 linked nucleosides. In certain embodiments, the 3′-wing of agapmer consists of 1 or 2 linked nucleosides. In certain embodiments,the 3′-wing of a gapmer consists of 2 to 4 linked nucleosides. Incertain embodiments, the 3′-wing of a gapmer consists of 2 or 3 linkednucleosides. In certain embodiments, the 3′-wing of a gapmer consists of3 or 4 linked nucleosides. In certain embodiments, the 3′-wing of agapmer consists of 1 nucleoside. In certain embodiments, the 3′-wing ofa gapmer consists of 2 linked nucleosides. In certain embodiments, the3′-wing of a gapmer consists of 3 linked nucleosides. In certainembodiments, the 3′-wing of a gapmer consists of 4 linked nucleosides.In certain embodiments, the 3′-wing of a gapmer consists of 5 linkednucleosides. In certain embodiments, the 3′-wing of a gapmer consists of6 linked nucleosides.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside. In certain embodiments, the 3′-wing of a gapmercomprises at least one constrained ethyl nucleoside. In certainembodiments, the 3′-wing of a gapmer comprises at least one LNAnucleoside. In certain embodiments, each nucleoside of the 3′-wing of agapmer is a bicyclic nucleoside. In certain embodiments, each nucleosideof the 3′-wing of a gapmer is a constrained ethyl nucleoside. In certainembodiments, each nucleoside of the 3′-wing of a gapmer is a LNAnucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onenon-bicyclic modified nucleoside. In certain embodiments, the 3′-wing ofa gapmer comprises at least two non-bicyclic modified nucleosides. Incertain embodiments, the 3′-wing of a gapmer comprises at least threenon-bicyclic modified nucleosides. In certain embodiments, the 3′-wingof a gapmer comprises at least four non-bicyclic modified nucleosides.In certain embodiments, the 3′-wing of a gapmer comprises at least one2′-substituted nucleoside. In certain embodiments, the 3′-wing of agapmer comprises at least one 2′-MOE nucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one 2′-OMe nucleoside. Incertain embodiments, each nucleoside of the 3′-wing of a gapmer is anon-bicyclic modified nucleoside. In certain embodiments, eachnucleoside of the 3′-wing of a gapmer is a 2′-substituted nucleoside. Incertain embodiments, each nucleoside of the 3′-wing of a gapmer is a2′-MOE nucleoside. In certain embodiments, each nucleoside of the3′-wing of a gapmer is a 2′-OMe nucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least one2′-deoxynucleoside. In certain embodiments, each nucleoside of the3′-wing of a gapmer is a 2′-deoxynucleoside. In a certain embodiments,the 3′-wing of a gapmer comprises at least one ribonucleoside. Incertain embodiments, each nucleoside of the 3′-wing of a gapmer is aribonucleoside. In certain embodiments, one, more than one, or each ofthe nucleosides of the 5′-wing is an RNA-like nucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one non-bicyclic modified nucleoside.In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one 2′-substituted nucleoside. Incertain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside and at least one 2′-MOE nucleoside. In certainembodiments, the 3′-wing of a gapmer comprises at least one bicyclicnucleoside and at least one 2′-OMe nucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one bicyclic nucleoside andat least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least oneconstrained ethyl nucleoside and at least one non-bicyclic modifiednucleoside. In certain embodiments, the 3′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-substitutednucleoside. In certain embodiments, the 3′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-MOEnucleoside. In certain embodiments, the 3′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one 2′-OMenucleoside. In certain embodiments, the 3′-wing of a gapmer comprises atleast one constrained ethyl nucleoside and at least one2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least oneLNA nucleoside and at least one non-bicyclic modified nucleoside. Incertain embodiments, the 3′-wing of a gapmer comprises at least one LNAnucleoside and at least one 2′-substituted nucleoside. In certainembodiments, the 3′-wing of a gapmer comprises at least one LNAnucleoside and at least one 2′-MOE nucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one LNA nucleoside and atleast one 2′-OMe nucleoside. In certain embodiments, the 3′-wing of agapmer comprises at least one LNA nucleoside and at least one2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside, at least one non-bicyclic modified nucleoside, andat least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing ofa gapmer comprises at least one constrained ethyl nucleoside, at leastone non-bicyclic modified nucleoside, and at least one2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmercomprises at least one LNA nucleoside, at least one non-bicyclicmodified nucleoside, and at least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside, at least one 2′-substituted nucleoside, and atleast one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of agapmer comprises at least one constrained ethyl nucleoside, at least one2′-substituted nucleoside, and at least one 2′-deoxynucleoside. Incertain embodiments, the 3′-wing of a gapmer comprises at least one LNAnucleoside, at least one 2′-substituted nucleoside, and at least one2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside, at least one 2′-MOE nucleoside, and at least one2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmercomprises at least one constrained ethyl nucleoside, at least one 2′-MOEnucleoside, and at least one 2′-deoxynucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one LNA nucleoside, at leastone 2′-MOE nucleoside, and at least one 2′-deoxynucleoside.

In certain embodiments, the 3′-wing of a gapmer comprises at least onebicyclic nucleoside, at least one 2′-OMe nucleoside, and at least one2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmercomprises at least one constrained ethyl nucleoside, at least one 2′-OMenucleoside, and at least one 2′-deoxynucleoside. In certain embodiments,the 3′-wing of a gapmer comprises at least one LNA nucleoside, at leastone 2′-OMe nucleoside, and at least one 2′-deoxynucleoside.

iii. Certain Central Regions (Gaps)

In certain embodiments, the gap of a gapmer consists of 6 to 20 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 6to 15 linked nucleosides. In certain embodiments, the gap of a gapmerconsists of 6 to 12 linked nucleosides. In certain embodiments, the gapof a gapmer consists of 6 to 10 linked nucleosides. In certainembodiments, the gap of a gapmer consists of 6 to 9 linked nucleosides.In certain embodiments, the gap of a gapmer consists of 6 to 8 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 6or 7 linked nucleosides. In certain embodiments, the gap of a gapmerconsists of 7 to 10 linked nucleosides. In certain embodiments, the gapof a gapmer consists of 7 to 9 linked nucleosides. In certainembodiments, the gap of a gapmer consists of 7 or 8 linked nucleosides.In certain embodiments, the gap of a gapmer consists of 8 to 10 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 8or 9 linked nucleosides. In certain embodiments, the gap of a gapmerconsists of 6 linked nucleosides. In certain embodiments, the gap of agapmer consists of 7 linked nucleosides. In certain embodiments, the gapof a gapmer consists of 8 linked nucleosides. In certain embodiments,the gap of a gapmer consists of 9 linked nucleosides. In certainembodiments, the gap of a gapmer consists of 10 linked nucleosides. Incertain embodiments, the gap of a gapmer consists of 11 linkednucleosides. In certain embodiments, the gap of a gapmer consists of 12linked nucleosides.

In certain embodiments, each nucleoside of the gap of a gapmer is a2′-deoxynucleoside. In certain embodiments, the gap comprises one ormore modified nucleosides. In certain embodiments, each nucleoside ofthe gap of a gapmer is a 2′-deoxynucleoside or is a modified nucleosidethat is “DNA-like.” In such embodiments, “DNA-like” means that thenucleoside has similar characteristics to DNA, such that a duplexcomprising the gapmer and an RNA molecule is capable of activating RNaseH. For example, under certain conditions, 2′-(ara)-F have been shown tosupport RNase H activation, and thus is DNA-like. In certainembodiments, one or more nucleosides of the gap of a gapmer is not a2′-deoxynucleoside and is not DNA-like. In certain such embodiments, thegapmer nonetheless supports RNase H activation (e.g., by virtue of thenumber or placement of the non-DNA nucleosides).

In certain embodiments, gaps comprise a stretch of unmodified2′-deoxynucleoside interrupted by one or more modified nucleosides, thusresulting in three sub-regions (two stretches of one or more2′-deoxynucleosides and a stretch of one or more interrupting modifiednucleosides). In certain embodiments, no stretch of unmodified2′-deoxynucleosides is longer than 5, 6, or 7 nucleosides. In certainembodiments, such short stretches is achieved by using short gapregions. In certain embodiments, short stretches are achieved byinterrupting a longer gap region.

In certain embodiments, the gap comprises one or more modifiednucleosides. In certain embodiments, the gap comprises one or moremodified nucleosides selected from among cEt, FHNA, LNA, and2-thio-thymidine. In certain embodiments, the gap comprises one modifiednucleoside. In certain embodiments, the gap comprises a 5′-substitutedsugar moiety selected from among 5′-Me, and 5′-(R)-Me. In certainembodiments, the gap comprises two modified nucleosides. In certainembodiments, the gap comprises three modified nucleosides. In certainembodiments, the gap comprises four modified nucleosides. In certainembodiments, the gap comprises two or more modified nucleosides and eachmodified nucleoside is the same. In certain embodiments, the gapcomprises two or more modified nucleosides and each modified nucleosideis different.

In certain embodiments, the gap comprises one or more modified linkages.In certain embodiments, the gap comprises one or more methyl phosphonatelinkages. In certain embodiments the gap comprises two or more modifiedlinkages. In certain embodiments, the gap comprises one or more modifiedlinkages and one or more modified nucleosides. In certain embodiments,the gap comprises one modified linkage and one modified nucleoside. Incertain embodiments, the gap comprises two modified linkages and two ormore modified nucleosides.

b. Certain Internucleoside Linkage Motifs

In certain embodiments, oligonucleotides comprise modifiedinternucleoside linkages arranged along the oligonucleotide or regionthereof in a defined pattern or modified internucleoside linkage motif.In certain embodiments, oligonucleotides comprise a region having analternating internucleoside linkage motif. In certain embodiments,oligonucleotides of the present disclosure comprise a region ofuniformly modified internucleoside linkages. In certain suchembodiments, the oligonucleotide comprises a region that is uniformlylinked by phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide is uniformly linked by phosphorothioateinternucleoside linkages. In certain embodiments, each internucleosidelinkage of the oligonucleotide is selected from phosphodiester andphosphorothioate. In certain embodiments, each internucleoside linkageof the oligonucleotide is selected from phosphodiester andphosphorothioate and at least one internucleoside linkage isphosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 7 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises at least8 phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 9 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises at least10 phosphorothioate internucleoside linkages. In certain embodiments,the oligonucleotide comprises at least 11 phosphorothioateinternucleoside linkages. In certain embodiments, the oligonucleotidecomprises at least 12 phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least 13phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 14 phosphorothioate internucleosidelinkages.

In certain embodiments, the oligonucleotide comprises at least one blockof at least 6 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 7 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 8 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 9 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 10 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least block of atleast one 12 consecutive phosphorothioate internucleoside linkages. Incertain such embodiments, at least one such block is located at the 3′end of the oligonucleotide. In certain such embodiments, at least onesuch block is located within 3 nucleosides of the 3′ end of theoligonucleotide. In certain embodiments, the oligonucleotide comprisesless than 15 phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide comprises less than 14 phosphorothioateinternucleoside linkages. In certain embodiments, the oligonucleotidecomprises less than 13 phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises less than 12phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises less than 11 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises lessthan 10 phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide comprises less than 9 phosphorothioateinternucleoside linkages. In certain embodiments, the oligonucleotidecomprises less than 8 phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises less than 7phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises less than 6 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises lessthan 5 phosphorothioate internucleoside linkages.

c. Certain Nucleobase Modification Motifs

In certain embodiments, oligonucleotides comprise chemical modificationsto nucleobases arranged along the oligonucleotide or region thereof in adefined pattern or nucleobases modification motif. In certain suchembodiments, nucleobase modifications are arranged in a gapped motif. Incertain embodiments, nucleobase modifications are arranged in analternating motif. In certain embodiments, each nucleobase is modified.In certain embodiments, none of the nucleobases is chemically modified.

In certain embodiments, oligonucleotides comprise a block of modifiednucleobases. In certain such embodiments, the block is at the 3′-end ofthe oligonucleotide. In certain embodiments the block is within 3nucleotides of the 3′-end of the oligonucleotide. In certain suchembodiments, the block is at the 5′-end of the oligonucleotide. Incertain embodiments the block is within 3 nucleotides of the 5′-end ofthe oligonucleotide.

In certain embodiments, nucleobase modifications are a function of thenatural base at a particular position of an oligonucleotide. Forexample, in certain embodiments each purine or each pyrimidine in anoligonucleotide is modified. In certain embodiments, each adenine ismodified. In certain embodiments, each guanine is modified. In certainembodiments, each thymine is modified. In certain embodiments, eachcytosine is modified. In certain embodiments, each uracil is modified.

In certain embodiments, some, all, or none of the cytosine moieties inan oligonucleotide are 5-methyl cytosine moieties. Herein, 5-methylcytosine is not a “modified nucleobase.” Accordingly, unless otherwiseindicated, unmodified nucleobases include both cytosine residues havinga 5-methyl and those lacking a 5 methyl. In certain embodiments, themethylation state of all or some cytosine nucleobases is specified.

In certain embodiments, chemical modifications to nucleobases compriseattachment of certain conjugate groups to nucleobases. In certainembodiments, each purine or each pyrimidine in an oligonucleotide may beoptionally modified to comprise a conjugate group.

d. Certain Overall Lengths

In certain embodiments, the present disclosure provides oligonucleotidesof any of a variety of ranges of lengths. In certain embodiments,oligonucleotides consist of X to Y linked nucleosides, where Xrepresents the fewest number of nucleosides in the range and Yrepresents the largest number of nucleosides in the range. In certainsuch embodiments, X and Y are each independently selected from 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, and 50; provided that X≤Y. For example, in certainembodiments, the oligonucleotide may consist of 8 to 9, 8 to 10, 8 to11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to19, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to27, 8 to 28, 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to30, 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to24, 10 to 25, 10 to 26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to12, 11 to 13, 11 to 14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to19, 11 to 20, 11 to 21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to26, 11 to 27, 11 to 28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to30 linked nucleosides. In embodiments where the number of nucleosides ofan oligonucleotide of a compound is limited, whether to a range or to aspecific number, the compound may, nonetheless further compriseadditional other substituents. For example, an oligonucleotidecomprising 8-30 nucleosides excludes oligonucleotides having 31nucleosides, but, unless otherwise indicated, such an oligonucleotidemay further comprise, for example one or more conjugate groups, terminalgroups, or other substituents.

Further, where an oligonucleotide is described by an overall lengthrange and by regions having specified lengths, and where the sum ofspecified lengths of the regions is less than the upper limit of theoverall length range, the oligonucleotide may have additionalnucleosides, beyond those of the specified regions, provided that thetotal number of nucleosides does not exceed the upper limit of theoverall length range.

5. Certain Antisense Oligonucleotide Chemistry Motifs

In certain embodiments, the chemical structural features of antisenseoligonucleotides are characterized by their sugar motif, internucleosidelinkage motif, nucleobase modification motif and overall length. Incertain embodiments, such parameters are each independent of oneanother. Thus, each internucleoside linkage of an oligonucleotide havinga gapmer sugar motif may be modified or unmodified and may or may notfollow the gapmer modification pattern of the sugar modifications. Thus,the internucleoside linkages within the wing regions of a sugar-gapmermay be the same or different from one another and may be the same ordifferent from the internucleoside linkages of the gap region. Likewise,such sugar-gapmer oligonucleotides may comprise one or more modifiednucleobase independent of the gapmer pattern of the sugar modifications.One of skill in the art will appreciate that such motifs may be combinedto create a variety of oligonucleotides.

In certain embodiments, the selection of internucleoside linkage andnucleoside modification are not independent of one another.

i. Certain Sequences and Targets

In certain embodiments, the invention provides antisenseoligonucleotides having a sequence complementary to a target nucleicacid. Such antisense compounds are capable of hybridizing to a targetnucleic acid, resulting in at least one antisense activity. In certainembodiments, antisense compounds specifically hybridize to one or moretarget nucleic acid. In certain embodiments, a specifically hybridizingantisense compound has a nucleobase sequence comprising a region havingsufficient complementarity to a target nucleic acid to allowhybridization and result in antisense activity and insufficientcomplementarity to any non-target so as to avoid or reduce non-specifichybridization to non-target nucleic acid sequences under conditions inwhich specific hybridization is desired (e.g., under physiologicalconditions for in vivo or therapeutic uses, and under conditions inwhich assays are performed in the case of in vitro assays). In certainembodiments, oligonucleotides are selective between a target andnon-target, even though both target and non-target comprise the targetsequence. In such embodiments, selectivity may result from relativeaccessibility of the target region of one nucleic acid molecule comparedto the other.

In certain embodiments, the present disclosure provides antisensecompounds comprising oligonucleotides that are fully complementary tothe target nucleic acid over the entire length of the oligonucleotide.In certain embodiments, oligonucleotides are 99% complementary to thetarget nucleic acid. In certain embodiments, oligonucleotides are 95%complementary to the target nucleic acid. In certain embodiments, sucholigonucleotides are 90% complementary to the target nucleic acid.

In certain embodiments, such oligonucleotides are 85% complementary tothe target nucleic acid. In certain embodiments, such oligonucleotidesare 80% complementary to the target nucleic acid. In certainembodiments, an antisense compound comprises a region that is fullycomplementary to a target nucleic acid and is at least 80% complementaryto the target nucleic acid over the entire length of theoligonucleotide. In certain such embodiments, the region of fullcomplementarity is from 6 to 14 nucleobases in length.

In certain embodiments, oligonucleotides comprise a hybridizing regionand a terminal region. In certain such embodiments, the hybridizingregion consists of 12-30 linked nucleosides and is fully complementaryto the target nucleic acid. In certain embodiments, the hybridizingregion includes one mismatch relative to the target nucleic acid. Incertain embodiments, the hybridizing region includes two mismatchesrelative to the target nucleic acid. In certain embodiments, thehybridizing region includes three mismatches relative to the targetnucleic acid. In certain embodiments, the terminal region consists of1-4 terminal nucleosides. In certain embodiments, the terminalnucleosides are at the 3′ end. In certain embodiments, one or more ofthe terminal nucleosides are not complementary to the target nucleicacid.

Antisense mechanisms include any mechanism involving the hybridizationof an oligonucleotide with target nucleic acid, wherein thehybridization results in a biological effect. In certain embodiments,such hybridization results in either target nucleic acid degradation oroccupancy with concomitant inhibition or stimulation of the cellularmachinery involving, for example, translation, transcription, orsplicing of the target nucleic acid.

One type of antisense mechanism involving degradation of target RNA isRNase H mediated antisense. RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. It is known in the art thatsingle-stranded antisense compounds which are “DNA-like” elicit RNase Hactivity in mammalian cells. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof DNA-like oligonucleotide-mediated inhibition of gene expression.

In certain embodiments, a conjugate group comprises a cleavable moiety.In certain embodiments, a conjugate group comprises one or morecleavable bond. In certain embodiments, a conjugate group comprises alinker. In certain embodiments, a linker comprises a protein bindingmoiety. In certain embodiments, a conjugate group comprises acell-targeting moiety (also referred to as a cell-targeting group). Incertain embodiments a cell-targeting moiety comprises a branching group.In certain embodiments, a cell-targeting moiety comprises one or moretethers. In certain embodiments, a cell-targeting moiety comprises acarbohydrate or carbohydrate cluster.

ii. Certain Cleavable Moieties

In certain embodiments, a cleavable moiety is a cleavable bond. Incertain embodiments, a cleavable moiety comprises a cleavable bond. Incertain embodiments, the conjugate group comprises a cleavable moiety.In certain such embodiments, the cleavable moiety attaches to theantisense oligonucleotide. In certain such embodiments, the cleavablemoiety attaches directly to the cell-targeting moiety. In certain suchembodiments, the cleavable moiety attaches to the conjugate linker. Incertain embodiments, the cleavable moiety comprises a phosphate orphosphodiester. In certain embodiments, the cleavable moiety is acleavable nucleoside or nucleoside analog. In certain embodiments, thenucleoside or nucleoside analog comprises an optionally protectedheterocyclic base selected from a purine, substituted purine, pyrimidineor substituted pyrimidine. In certain embodiments, the cleavable moietyis a nucleoside comprising an optionally protected heterocyclic baseselected from uracil, thymine, cytosine, 4-N-benzoylcytosine,5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine,6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. In certainembodiments, the cleavable moiety is 2′-deoxy nucleoside that isattached to the 3′ position of the antisense oligonucleotide by aphosphodiester linkage and is attached to the linker by a phosphodiesteror phosphorothioate linkage. In certain embodiments, the cleavablemoiety is 2′-deoxy adenosine that is attached to the 3′ position of theantisense oligonucleotide by a phosphodiester linkage and is attached tothe linker by a phosphodiester or phosphorothioate linkage. In certainembodiments, the cleavable moiety is 2′-deoxy adenosine that is attachedto the 3′ position of the antisense oligonucleotide by a phosphodiesterlinkage and is attached to the linker by a phosphodiester linkage.

In certain embodiments, the cleavable moiety is attached to the 3′position of the antisense oligonucleotide. In certain embodiments, thecleavable moiety is attached to the 5′ position of the antisenseoligonucleotide. In certain embodiments, the cleavable moiety isattached to a 2′ position of the antisense oligonucleotide. In certainembodiments, the cleavable moiety is attached to the antisenseoligonucleotide by a phosphodiester linkage. In certain embodiments, thecleavable moiety is attached to the linker by either a phosphodiester ora phosphorothioate linkage. In certain embodiments, the cleavable moietyis attached to the linker by a phosphodiester linkage. In certainembodiments, the conjugate group does not include a cleavable moiety.

In certain embodiments, the cleavable moiety is cleaved after thecomplex has been administered to an animal only after being internalizedby a targeted cell. Inside the cell the cleavable moiety is cleavedthereby releasing the active antisense oligonucleotide. While notwanting to be bound by theory it is believed that the cleavable moietyis cleaved by one or more nucleases within the cell. In certainembodiments, the one or more nucleases cleave the phosphodiester linkagebetween the cleavable moiety and the linker. In certain embodiments, thecleavable moiety has a structure selected from among the following:

wherein each of Bx, Bx₁, Bx₂, and Bx₃ is independently a heterocyclicbase moiety. In certain embodiments, the cleavable moiety has astructure selected from among the following:

iii. Certain Linkers

In certain embodiments, the conjugate groups comprise a linker. Incertain such embodiments, the linker is covalently bound to thecleavable moiety. In certain such embodiments, the linker is covalentlybound to the antisense oligonucleotide. In certain embodiments, thelinker is covalently bound to a cell-targeting moiety. In certainembodiments, the linker further comprises a covalent attachment to asolid support. In certain embodiments, the linker further comprises acovalent attachment to a protein binding moiety. In certain embodiments,the linker further comprises a covalent attachment to a solid supportand further comprises a covalent attachment to a protein binding moiety.In certain embodiments, the linker includes multiple positions forattachment of tethered ligands. In certain embodiments, the linkerincludes multiple positions for attachment of tethered ligands and isnot attached to a branching group. In certain embodiments, the linkerfurther comprises one or more cleavable bond. In certain embodiments,the conjugate group does not include a linker.

In certain embodiments, the linker includes at least a linear groupcomprising groups selected from alkyl, amide, disulfide, polyethyleneglycol, ether, thioether (—S—) and hydroxylamino (—O—N(H)—) groups. Incertain embodiments, the linear group comprises groups selected fromalkyl, amide and ether groups. In certain embodiments, the linear groupcomprises groups selected from alkyl and ether groups. In certainembodiments, the linear group comprises at least one phosphorus linkinggroup. In certain embodiments, the linear group comprises at least onephosphodiester group. In certain embodiments, the linear group includesat least one neutral linking group. In certain embodiments, the lineargroup is covalently attached to the cell-targeting moiety and thecleavable moiety. In certain embodiments, the linear group is covalentlyattached to the cell-targeting moiety and the antisense oligonucleotide.In certain embodiments, the linear group is covalently attached to thecell-targeting moiety, the cleavable moiety and a solid support. Incertain embodiments, the linear group is covalently attached to thecell-targeting moiety, the cleavable moiety, a solid support and aprotein binding moiety. In certain embodiments, the linear groupincludes one or more cleavable bond.

In certain embodiments, the linker includes the linear group covalentlyattached to a scaffold group. In certain embodiments, the scaffoldincludes a branched aliphatic group comprising groups selected fromalkyl, amide, disulfide, polyethylene glycol, ether, thioether andhydroxylamino groups. In certain embodiments, the scaffold includes abranched aliphatic group comprising groups selected from alkyl, amideand ether groups. In certain embodiments, the scaffold includes at leastone mono or polycyclic ring system. In certain embodiments, the scaffoldincludes at least two mono or polycyclic ring systems. In certainembodiments, the linear group is covalently attached to the scaffoldgroup and the scaffold group is covalently attached to the cleavablemoiety and the linker. In certain embodiments, the linear group iscovalently attached to the scaffold group and the scaffold group iscovalently attached to the cleavable moiety, the linker and a solidsupport. In certain embodiments, the linear group is covalently attachedto the scaffold group and the scaffold group is covalently attached tothe cleavable moiety, the linker and a protein binding moiety. Incertain embodiments, the linear group is covalently attached to thescaffold group and the scaffold group is covalently attached to thecleavable moiety, the linker, a protein binding moiety and a solidsupport. In certain embodiments, the scaffold group includes one or morecleavable bond.

In certain embodiments, the linker includes a protein binding moiety. Incertain embodiments, the protein binding moiety is a lipid such as forexample including but not limited to cholesterol, cholic acid,adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine), a vitamin (e.g., folate, vitamin A,vitamin E, biotin, pyridoxal), a peptide, a carbohydrate (e.g.,monosaccharide, disaccharide, trisaccharide, tetrasaccharide,oligosaccharide, polysaccharide), an endosomolytic component, a steroid(e.g., uvaol, hecigenin, diosgenin), a terpene (e.g., triterpene, e.g.,sarsasapogenin, friedelin, epifriedelanol derivatized lithocholic acid),or a cationic lipid. In certain embodiments, the protein binding moietyis a C16 to C22 long chain saturated or unsaturated fatty acid,cholesterol, cholic acid, vitamin E, adamantane or 1-pentafluoropropyl.

In certain embodiments, a linker has a structure selected from among:

wherein each n is, independently, from 1 to 20; and p is from 1 to 6.

In certain embodiments, a linker has a structure selected from among:

wherein each n is, independently, from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

wherein n is from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

wherein each L is, independently, a phosphorus linking group or aneutral linking group; and

each n is, independently, from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

wherein n is from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, the conjugate linker has the structure:

In certain embodiments, the conjugate linker has the structure:

In certain embodiments, a linker has a structure selected from among:

In certain embodiments, a linker has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

iv. Certain Cell-Targeting Moieties

In certain embodiments, conjugate groups comprise cell-targetingmoieties. Certain such cell-targeting moieties increase cellular uptakeof antisense compounds. In certain embodiments, cell-targeting moietiescomprise a branching group, one or more tether, and one or more ligand.In certain embodiments, cell-targeting moieties comprise a branchinggroup, one or more tether, one or more ligand and one or more cleavablebond.

1. Certain Branching Groups

In certain embodiments, the conjugate groups comprise a targeting moietycomprising a branching group and at least two tethered ligands. Incertain embodiments, the branching group attaches the conjugate linker.In certain embodiments, the branching group attaches the cleavablemoiety. In certain embodiments, the branching group attaches theantisense oligonucleotide. In certain embodiments, the branching groupis covalently attached to the linker and each of the tethered ligands.In certain embodiments, the branching group comprises a branchedaliphatic group comprising groups selected from alkyl, amide, disulfide,polyethylene glycol, ether, thioether and hydroxylamino groups. Incertain embodiments, the branching group comprises groups selected fromalkyl, amide and ether groups. In certain embodiments, the branchinggroup comprises groups selected from alkyl and ether groups. In certainembodiments, the branching group comprises a mono or polycyclic ringsystem. In certain embodiments, the branching group comprises one ormore cleavable bond. In certain embodiments, the conjugate group doesnot include a branching group.

In certain embodiments, a branching group has a structure selected fromamong:

wherein each n is, independently, from 1 to 20;

j is from 1 to 3; and

m is from 2 to 6.

In certain embodiments, a branching group has a structure selected fromamong:

wherein each n is, independently, from 1 to 20; and

m is from 2 to 6.

In certain embodiments, a branching group has a structure selected fromamong:

In certain embodiments, a branching group has a structure selected fromamong:

wherein each A₁ is independently, O, S, C═O or NH; and

each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected fromamong:

wherein each A₁ is independently, O, S, C═O or NH; and

each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected fromamong:

wherein A₁ is O, S, C═O or NH; and

each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected fromamong:

In certain embodiments, a branching group has a structure selected fromamong:

In certain embodiments, a branching group has a structure selected fromamong:

2. Certain Tethers

In certain embodiments, conjugate groups comprise one or more tetherscovalently attached to the branching group. In certain embodiments,conjugate groups comprise one or more tethers covalently attached to thelinking group. In certain embodiments, each tether is a linear aliphaticgroup comprising one or more groups selected from alkyl, ether,thioether, disulfide, amide and polyethylene glycol groups in anycombination. In certain embodiments, each tether is a linear aliphaticgroup comprising one or more groups selected from alkyl, substitutedalkyl, ether, thioether, disulfide, amide, phosphodiester andpolyethylene glycol groups in any combination. In certain embodiments,each tether is a linear aliphatic group comprising one or more groupsselected from alkyl, ether and amide groups in any combination. Incertain embodiments, each tether is a linear aliphatic group comprisingone or more groups selected from alkyl, substituted alkyl,phosphodiester, ether and amide groups in any combination. In certainembodiments, each tether is a linear aliphatic group comprising one ormore groups selected from alkyl and phosphodiester in any combination.In certain embodiments, each tether comprises at least one phosphoruslinking group or neutral linking group.

In certain embodiments, the tether includes one or more cleavable bond.In certain embodiments, the tether is attached to the branching groupthrough either an amide or an ether group. In certain embodiments, thetether is attached to the branching group through a phosphodiestergroup. In certain embodiments, the tether is attached to the branchinggroup through a phosphorus linking group or neutral linking group. Incertain embodiments, the tether is attached to the branching groupthrough an ether group. In certain embodiments, the tether is attachedto the ligand through either an amide or an ether group. In certainembodiments, the tether is attached to the ligand through an ethergroup. In certain embodiments, the tether is attached to the ligandthrough either an amide or an ether group. In certain embodiments, thetether is attached to the ligand through an ether group.

In certain embodiments, each tether comprises from about 8 to about 20atoms in chain length between the ligand and the branching group. Incertain embodiments, each tether group comprises from about 10 to about18 atoms in chain length between the ligand and the branching group. Incertain embodiments, each tether group comprises about 13 atoms in chainlength.

In certain embodiments, a tether has a structure selected from among:

wherein each n is, independently, from 1 to 20; and

each p is from 1 to about 6.

In certain embodiments, a tether has a structure selected from among:

In certain embodiments, a tether has a structure selected from among:

-   -   wherein each n is, independently, from 1 to 20.

In certain embodiments, a tether has a structure selected from among:

-   -   wherein L is either a phosphorus linking group or a neutral        linking group;    -   Z₁ is C(═O)O—R₂;    -   Z₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky;    -   R₂ is H, C₁-C₆ alkyl or substituted C₁-C₆ alky; and    -   each m₁ is, independently, from 0 to 20 wherein at least one m₁        is greater than 0 for each tether.

In certain embodiments, a tether has a structure selected from among:

In certain embodiments, a tether has a structure selected from among:

-   -   wherein Z₂ is H or CH₃; and    -   each m₁ is, independently, from 0 to 20 wherein at least one m₁        is greater than 0 for each tether.

In certain embodiments, a tether has a structure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

In certain embodiments, a tether comprises a phosphorus linking group.In certain embodiments, a tether does not comprise any amide bonds. Incertain embodiments, a tether comprises a phosphorus linking group anddoes not comprise any amide bonds.

3. Certain Ligands

In certain embodiments, the present disclosure provides ligands whereineach ligand is covalently attached to a tether. In certain embodiments,each ligand is selected to have an affinity for at least one type ofreceptor on a target cell. In certain embodiments, ligands are selectedthat have an affinity for at least one type of receptor on the surfaceof a mammalian liver cell. In certain embodiments, ligands are selectedthat have an affinity for the hepatic asialoglycoprotein receptor(ASGP-R). In certain embodiments, each ligand is a carbohydrate. Incertain embodiments, each ligand is, independently selected fromgalactose, N-acetyl galactoseamine, mannose, glucose, glucosamone andfucose. In certain embodiments, each ligand is N-acetyl galactoseamine(GalNAc). In certain embodiments, the targeting moiety comprises 2 to 6ligands. In certain embodiments, the targeting moiety comprises 3ligands. In certain embodiments, the targeting moiety comprises 3N-acetyl galactoseamine ligands.

In certain embodiments, the ligand is a carbohydrate, carbohydratederivative, modified carbohydrate, multivalent carbohydrate cluster,polysaccharide, modified polysaccharide, or polysaccharide derivative.In certain embodiments, the ligand is an amino sugar or a thio sugar.For example, amino sugars may be selected from any number of compoundsknown in the art, for example glucosamine, sialic acid,α-D-galactosamine, N-Acetylgalactosamine,2-acetamido-2-deoxy-D-galactopyranose (GalNAc),2-Amino-3-O—[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose (β-muramicacid), 2-Deoxy-2-methylamino-L-glucopyranose,4,6-Dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose,2-Deoxy-2-sulfoamino-D-glucopyranose and N-sulfo-D-glucosamine, andN-Glycoloyl-α-neuraminic acid. For example, thio sugars may be selectedfrom the group consisting of 5-Thio-β-D-glucopyranose, Methyl2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside,4-Thio-β-D-galactopyranose, and ethyl3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside.

In certain embodiments, “GalNac” or “Gal-NAc” refers to2-(Acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in theliterature as N-acetyl galactosamine. In certain embodiments, “N-acetylgalactosamine” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose. Incertain embodiments, “GalNac” or “Gal-NAc” refers to2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments,“GalNac” or “Gal-NAc” refers to2-(Acetylamino)-2-deoxy-D-galactopyranose, which includes both the(3-form: 2-(Acetylamino)-2-deoxy-β-D-galactopyranose and α-form:2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, boththe β-form: 2-(Acetylamino)-2-deoxy-β-D-galactopyranose and α-form:2-(Acetylamino)-2-deoxy-D-galactopyranose may be used interchangeably.Accordingly, in structures in which one form is depicted, thesestructures are intended to include the other form as well. For example,where the structure for an α-form:2-(Acetylamino)-2-deoxy-D-galactopyranose is shown, this structure isintended to include the other form as well. In certain embodiments, Incertain preferred embodiments, the β-form2-(Acetylamino)-2-deoxy-D-galactopyranose is the preferred embodiment.

In certain embodiments one or more ligand has a structure selected fromamong:

wherein each R₁ is selected from OH and NHCOOH.

In certain embodiments one or more ligand has a structure selected fromamong:

In certain embodiments one or more ligand has a structure selected fromamong:

In certain embodiments one or more ligand has a structure selected fromamong:

i. Certain Conjugates

In certain embodiments, conjugate groups comprise the structuralfeatures above. In certain such embodiments, conjugate groups have thefollowing structure:

wherein each n is, independently, from 1 to 20.

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

wherein each n is, independently, from 1 to 20;

Z is H or a linked solid support;

Q is an antisense compound;

X is O or S; and

Bx is a heterocyclic base moiety.

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain embodiments, conjugates do not comprise a pyrrolidine.

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain such embodiments, conjugate groups have the followingstructure:

In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein X is a substituted or unsubstituted tether of six to elevenconsecutively bonded atoms.In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein X is a substituted or unsubstituted tether of ten consecutivelybonded atoms.In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein X is a substituted or unsubstituted tether of four to elevenconsecutively bonded atoms and wherein the tether comprises exactly oneamide bond.In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein Y and Z are independently selected from a C₁-C₁₂ substituted orunsubstituted alkyl, alkenyl, or alkynyl group, or a group comprising anether, a ketone, an amide, an ester, a carbamate, an amine, apiperidine, a phosphate, a phosphodiester, a phosphorothioate, atriazole, a pyrrolidine, a disulfide, or a thioether.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

wherein Y and Z are independently selected from a C₁-C₁₂ substituted orunsubstituted alkyl group, or a group comprising exactly one ether orexactly two ethers, an amide, an amine, a piperidine, a phosphate, aphosphodiester, or a phosphorothioate.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

wherein Y and Z are independently selected from a C₁-C₁₂ substituted orunsubstituted alkyl group.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

wherein m and n are independently selected from 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, and 12.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

wherein m is 4, 5, 6, 7, or 8, and n is 1, 2, 3, or 4.In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein X is a substituted or unsubstituted tether of four to thirteenconsecutively bonded atoms, and whereinX does not comprise an ether group.In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein X is a substituted or unsubstituted tether of eightconsecutively bonded atoms, and wherein X does not comprise an ethergroup.In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein X is a substituted or unsubstituted tether of four to thirteenconsecutively bonded atoms, and wherein the tether comprises exactly oneamide bond, and wherein X does not comprise an ether group.In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein X is a substituted or unsubstituted tether of four to thirteenconsecutively bonded atoms and wherein the tether consists of an amidebond and a substituted or unsubstituted C₂-C₁₁ alkyl group.In certain embodiments, the cell-targeting moiety of the conjugate grouphas the following structure:

wherein Y is selected from a C₁-C₁₂ substituted or unsubstituted alkyl,alkenyl, or alkynyl group, or a group comprising an ether, a ketone, anamide, an ester, a carbamate, an amine, a piperidine, a phosphate, aphosphodiester, a phosphorothioate, a triazole, a pyrrolidine, adisulfide, or a thioether.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

wherein Y is selected from a C₁-C₁₂ substituted or unsubstituted alkylgroup, or a group comprising an ether, an amine, a piperidine, aphosphate, a phosphodiester, or a phosphorothioate.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

wherein Y is selected from a C₁-C₁₂ substituted or unsubstituted alkylgroup.In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

Wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

In certain such embodiments, the cell-targeting moiety of the conjugategroup has the following structure:

wherein n is 4, 5, 6, 7, or 8.

b. Certain Conjugated Antisense Compounds

In certain embodiments, the conjugates are bound to a nucleoside of theantisense oligonucleotide at the 2′, 3′, of 5′ position of thenucleoside. In certain embodiments, a conjugated antisense compound hasthe following structure:

A-B-C-DE-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has thefollowing structure:

A-C-DE-F)_(q)

wherein

A is the antisense oligonucleotide;

C is the conjugate linker

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain such embodiments, the conjugate linker comprises at least onecleavable bond.

In certain such embodiments, the branching group comprises at least onecleavable bond.

In certain embodiments each tether comprises at least one cleavablebond.

In certain embodiments, the conjugates are bound to a nucleoside of theantisense oligonucleotide at the 2′, 3′, of 5′ position of thenucleoside.

In certain embodiments, a conjugated antisense compound has thefollowing structure:

A-B-CE-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

C is the conjugate linker

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, the conjugates are bound to a nucleoside of theantisense oligonucleotide at the 2′, 3′, of 5′ position of thenucleoside. In certain embodiments, a conjugated antisense compound hasthe following structure:

A-CE-F)_(q)

wherein

A is the antisense oligonucleotide;

C is the conjugate linker

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has thefollowing structure:

A-B-DE-F)_(q)

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has thefollowing structure:

A-DE-F)_(q)

wherein

A is the antisense oligonucleotide;

D is the branching group

each E is a tether;

each F is a ligand; and

q is an integer between 1 and 5.

In certain such embodiments, the conjugate linker comprises at least onecleavable bond.

In certain embodiments each tether comprises at least one cleavablebond.

In certain embodiments, a conjugated antisense compound has a structureselected from among the following:

In certain embodiments, a conjugated antisense compound has a structureselected from among the following:

In certain embodiments, a conjugated antisense compound has a structureselected from among the following:

In certain embodiments, the conjugated antisense compound has thefollowing structure:

Representative United States patents, United States patent applicationpublications, and international patent application publications thatteach the preparation of certain of the above noted conjugates,conjugated antisense compounds, tethers, linkers, branching groups,ligands, cleavable moieties as well as other modifications includewithout limitation, U.S. Pat. No. 5,994,517, U.S. Pat. No. 6,300,319,U.S. Pat. No. 6,660,720, U.S. Pat. No. 6,906,182, U.S. Pat. No.7,262,177, U.S. Pat. No. 7,491,805, U.S. Pat. No. 8,106,022, U.S. Pat.No. 7,723,509, US 2006/0148740, US 2011/0123520, WO 2013/033230 and WO2012/037254, each of which is incorporated by reference herein in itsentirety.

Representative publications that teach the preparation of certain of theabove noted conjugates, conjugated antisense compounds, tethers,linkers, branching groups, ligands, cleavable moieties as well as othermodifications include without limitation, BIESSEN et al., “TheCholesterol Derivative of a Triantennary Galactoside with High Affinityfor the Hepatic Asialoglycoprotein Receptor: a Potent CholesterolLowering Agent” J. Med. Chem. (1995) 38:1846-1852, BIESSEN et al.,“Synthesis of Cluster Galactosides with High Affinity for the HepaticAsialoglycoprotein Receptor” J. Med. Chem. (1995) 38:1538-1546, LEE etal., “New and more efficient multivalent glyco-ligands forasialoglycoprotein receptor of mammalian hepatocytes” Bioorganic &Medicinal Chemistry (2011) 19:2494-2500, RENSEN et al., “Determinationof the Upper Size Limit for Uptake and Processing of Ligands by theAsialoglycoprotein Receptor on Hepatocytes in Vitro and in Vivo” J.Biol. Chem. (2001) 276(40):37577-37584, RENSEN et al., “Design andSynthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids forTargeting of Lipoproteins to the Hepatic Asialoglycoprotein Receptor” J.Med. Chem. (2004) 47:5798-5808, SLIEDREGT et al., “Design and Synthesisof Novel Amphiphilic Dendritic Galactosides for Selective Targeting ofLiposomes to the Hepatic Asialoglycoprotein Receptor” J. Med. Chem.(1999) 42:609-618, and Valentijn et al., “Solid-phase synthesis oflysine-based cluster galactosides with high affinity for theAsialoglycoprotein Receptor” Tetrahedron, 1997, 53(2), 759-770, each ofwhich is incorporated by reference herein in its entirety.

In certain embodiments, conjugated antisense compounds comprise an RNaseH based oligonucleotide (such as a gapmer) or a splice modulatingoligonucleotide (such as a fully modified oligonucleotide) and anyconjugate group comprising at least one, two, or three GalNAc groups. Incertain embodiments a conjugated antisense compound comprises anyconjugate group found in any of the following references: Lee, CarbohydrRes, 1978, 67, 509-514; Connolly et al., J Biol Chem, 1982, 257,939-945; Pavia et al., Int J Pep Protein Res, 1983, 22, 539-548; Lee etal., Biochem, 1984, 23, 4255-4261; Lee et al., Glycoconjugate J, 1987,4, 317-328; Toyokuni et al., Tetrahedron Lett, 1990, 31, 2673-2676;Biessen et al., J Med Chem, 1995, 38, 1538-1546; Valentijn et al.,Tetrahedron, 1997, 53, 759-770; Kim et al., Tetrahedron Lett, 1997, 38,3487-3490; Lee et al., Bioconjug Chem, 1997, 8, 762-765; Kato et al.,Glycobiol, 2001, 11, 821-829; Rensen et al., J Biol Chem, 2001, 276,37577-37584; Lee et al., Methods Enzymol, 2003, 362, 38-43; Westerlindet al., Glycoconj J, 2004, 21, 227-241; Lee et al., Bioorg Med ChemLett, 2006, 16(19), 5132-5135; Maierhofer et al., Bioorg Med Chem, 2007,15, 7661-7676; Khorev et al., Bioorg Med Chem, 2008, 16, 5216-5231; Leeet al., Bioorg Med Chem, 2011, 19, 2494-2500; Kornilova et al., AnalytBiochem, 2012, 425, 43-46; Pujol et al., Angew Chemie Int Ed Engl, 2012,51, 7445-7448; Biessen et al., J Med Chem, 1995, 38, 1846-1852;Sliedregt et al., J Med Chem, 1999, 42, 609-618; Rensen et al., J MedChem, 2004, 47, 5798-5808; Rensen et al., Arterioscler Thromb Vasc Biol,2006, 26, 169-175; van Rossenberg et al., Gene Ther, 2004, 11, 457-464;Sato et al., J Am Chem Soc, 2004, 126, 14013-14022; Lee et al., J OrgChem, 2012, 77, 7564-7571; Biessen et al., FASEB J, 2000, 14, 1784-1792;Rajur et al., Bioconjug Chem, 1997, 8, 935-940; Duff et al., MethodsEnzymol, 2000, 313, 297-321; Maier et al., Bioconjug Chem, 2003, 14,18-29; Jayaprakash et al., Org Lett, 2010, 12, 5410-5413; Manoharan,Antisense Nucleic Acid Drug Dev, 2002, 12, 103-128; Merwin et al.,Bioconjug Chem, 1994, 5, 612-620; Tomiya et al., Bioorg Med Chem, 2013,21, 5275-5281; International applications WO1998/013381; WO2011/038356;WO1997/046098; WO2008/098788; WO2004/101619; WO2012/037254;WO2011/120053; WO2011/100131; WO2011/163121; WO2012/177947;WO2013/033230; WO2013/075035; WO2012/083185; WO2012/083046;WO2009/082607; WO2009/134487; WO2010/144740; WO2010/148013;WO1997/020563; WO2010/088537; WO2002/043771; WO2010/129709;WO2012/068187; WO2009/126933; WO2004/024757; WO2010/054406;WO2012/089352; WO2012/089602; WO2013/166121; WO2013/165816; U.S. Pat.Nos. 4,751,219; 8,552,163; 6,908,903; 7,262,177; 5,994,517; 6,300,319;8,106,022; 7,491,805; 7,491,805; 7,582,744; 8,137,695; 6,383,812;6,525,031; 6,660,720; 7,723,509; 8,541,548; 8,344,125; 8,313,772;8,349,308; 8,450,467; 8,501,930; 8,158,601; 7,262,177; 6,906,182;6,620,916; 8,435,491; 8,404,862; 7,851,615; Published U.S. PatentApplication Publications US2011/0097264; US2011/0097265; US2013/0004427;US2005/0164235; US2006/0148740; US2008/0281044; US2010/0240730;US2003/0119724; US2006/0183886; US2008/0206869; US2011/0269814;US2009/0286973; US2011/0207799; US2012/0136042; US2012/0165393;US2008/0281041; US2009/0203135; US2012/0035115; US2012/0095075;US2012/0101148; US2012/0128760; US2012/0157509; US2012/0230938;US2013/0109817; US2013/0121954; US2013/0178512; US2013/0236968;US2011/0123520; US2003/0077829; US2008/0108801; and US2009/0203132; eachof which is incorporated by reference in its entirety.

C. Certain Uses and Features

In certain embodiments, conjugated antisense compounds exhibit potenttarget RNA reduction in vivo. In certain embodiments, unconjugatedantisense compounds accumulate in the kidney. In certain embodiments,conjugated antisense compounds accumulate in the liver. In certainembodiments, conjugated antisense compounds are well tolerated. Suchproperties render conjugated antisense compounds particularly useful forinhibition of many target RNAs, including, but not limited to thoseinvolved in metabolic, cardiovascular and other diseases, disorders orconditions. Thus, provided herein are methods of treating such diseases,disorders or conditions by contacting liver tissues with the conjugatedantisense compounds targeted to RNAs associated with such diseases,disorders or conditions. Thus, also provided are methods forameliorating any of a variety of metabolic, cardiovascular and otherdiseases, disorders or conditions with the conjugated antisensecompounds of the present invention.

In certain embodiments, conjugated antisense compounds are more potentthan unconjugated counterpart at a particular tissue concentration.Without wishing to be bound by any theory or mechanism, in certainembodiments, the conjugate may allow the conjugated antisense compoundto enter the cell more efficiently or to enter the cell moreproductively. For example, in certain embodiments conjugated antisensecompounds may exhibit greater target reduction as compared to itsunconjugated counterpart wherein both the conjugated antisense compoundand its unconjugated counterpart are present in the tissue at the sameconcentrations. For example, in certain embodiments conjugated antisensecompounds may exhibit greater target reduction as compared to itsunconjugated counterpart wherein both the conjugated antisense compoundand its unconjugated counterpart are present in the liver at the sameconcentrations.

Productive and non-productive uptake of oligonucleotides has beeddiscussed previously (See e.g. Geary, R. S., E. Wancewicz, et al.(2009). “Effect of Dose and Plasma Concentration on Liver Uptake andPharmacologic Activity of a 2′-Methoxyethyl Modified Chimeric AntisenseOligonucleotide Targeting PTEN.” Biochem. Pharmacol. 78(3): 284-91; &Koller, E., T. M. Vincent, et al. (2011). “Mechanisms of single-strandedphosphorothioate modified antisense oligonucleotide accumulation inhepatocytes.” Nucleic Acids Res. 39(11): 4795-807). Conjugate groupsdescribed herein may improve productive uptake.

In certain embodiments, the conjugate groups described herein mayfurther improve potency by increasing the affinity of the conjugatedantisense compound for a particular type of cell or tissue. In certainembodiments, the conjugate groups described herein may further improvepotency by increasing recognition of the conjugated antisense compoundby one or more cell-surface receptors. In certain embodiments, theconjugate groups described herein may further improve potency byfacilitating endocytosis of the conjugated antisense compound.

In certain embodiments, the cleavable moiety may further improve potencyby allowing the conjugate to be cleaved from the antisenseoligonucleotide after the conjugated antisense compound has entered thecell. Accordingly, in certain embodiments, conjugated antisensecompounds can be administered at doses lower than would be necessary forunconjugated antisense oligonucleotides.

Phosphorothioate linkages have been incorporated into antisenseoligonucleotides previously. Such phosphorothioate linkages areresistant to nucleases and so improve stability of the oligonucleotide.Further, phosphorothioate linkages also bind certain proteins, whichresults in accumulation of antisense oligonucleotide in the liver.Oligonucleotides with fewer phosphorothioate linkages accumulate less inthe liver and more in the kidney (see, for example, Geary, R.,“Pharmacokinetic Properties of 2′-O-(2-Methoxyethyl)-ModifiedOligonucleotide Analogs in Rats,” Journal of Pharmacology andExperimental Therapeutics, Vol. 296, No. 3, 890-897; & PharmacologicalProperties of 2′-O-Methoxyethyl Modified Oligonucleotides in Antisense aDrug Technology, Chapter 10, Crooke, S. T., ed., 2008) In certainembodiments, oligonucleotides with fewer phosphorothioateinternucleoside linkages and more phosphodiester internucleosidelinkages accumulate less in the liver and more in the kidney. Whentreating diseases in the liver, this is undesirable for several reasons(1) less drug is getting to the site of desired action (liver); (2) drugis escaping into the urine; and (3) the kidney is exposed to relativelyhigh concentration of drug which can result in toxicities in the kidney.Thus, for liver diseases, phosphorothioate linkages provide importantbenefits.

In certain embodiments, however, administration of oligonucleotidesuniformly linked by phosphorothioate internucleoside linkages inducesone or more proinflammatory reactions. (see for example: J Lab Clin Med.1996 September; 128(3):329-38. “Amplification of antibody production byphosphorothioate oligodeoxynucleotides”. Branda et al.; and see also forexample: Toxicologic Properties in Antisense a Drug Technology, Chapter12, pages 342-351, Crooke, S. T., ed., 2008). In certain embodiments,administration of oligonucleotides wherein most of the internucleosidelinkages comprise phosphorothioate internucleoside linkages induces oneor more proinflammatory reactions.

In certain embodiments, the degree of proinflammatory effect may dependon several variables (e.g. backbone modification, off-target effects,nucleobase modifications, and/or nucleoside modifications) see forexample: Toxicologic Properties in Antisense a Drug Technology, Chapter12, pages 342-351, Crooke, S. T., ed., 2008). In certain embodiments,the degree of proinflammatory effect may be mitigated by adjusting oneor more variables. For example the degree of proinflammatory effect of agiven oligonucleotide may be mitigated by replacing any number ofphosphorothioate internucleoside linkages with phosphodiesterinternucleoside linkages and thereby reducing the total number ofphosphorothioate internucleoside linkages.

In certain embodiments, it would be desirable to reduce the number ofphosphorothioate linkages, if doing so could be done without losingstability and without shifting the distribution from liver to kidney.For example, in certain embodiments, the number of phosphorothioatelinkages may be reduced by replacing phosphorothioate linkages withphosphodiester linkages. In such an embodiment, the antisense compoundhaving fewer phosphorothioate linkages and more phosphodiester linkagesmay induce less proinflammatory reactions or no proinflammatoryreaction. Although the antisense compound having fewer phosphorothioatelinkages and more phosphodiester linkages may induce fewerproinflammatory reactions, the antisense compound having fewerphosphorothioate linkages and more phosphodiester linkages may notaccumulate in the liver and may be less efficacious at the same orsimilar dose as compared to an antisense compound having morephosphorothioate linkages. In certain embodiments, it is thereforedesirable to design an antisense compound that has a plurality ofphosphodiester bonds and a plurality of phosphorothioate bonds but whichalso possesses stability and good distribution to the liver.

In certain embodiments, conjugated antisense compounds accumulate morein the liver and less in the kidney than unconjugated counterparts, evenwhen some of the phosporothioate linkages are replaced with lessproinflammatory phosphodiester internucleoside linkages. In certainembodiments, conjugated antisense compounds accumulate more in the liverand are not excreted as much in the urine compared to its unconjugatedcounterparts, even when some of the phosporothioate linkages arereplaced with less proinflammatory phosphodiester internucleosidelinkages. In certain embodiments, the use of a conjugate allows one todesign more potent and better tolerated antisense drugs. Indeed, incertain embodiments, conjugated antisense compounds have largertherapeutic indexes than unconjugated counterparts. This allows theconjugated antisense compound to be administered at a higher absolutedose, because there is less risk of proinflammatory response and lessrisk of kidney toxicity. This higher dose, allows one to dose lessfrequently, since the clearance (metabolism) is expected to be similar.Further, because the compound is more potent, as described above, onecan allow the concentration to go lower before the next dose withoutlosing therapeutic activity, allowing for even longer periods betweendosing.

In certain embodiments, the inclusion of some phosphorothioate linkagesremains desirable. For example, the terminal linkages are vulnerable toexonucleases and so in certain embodiments, those linkages arephosphorothioate or other modified linkage. Internucleoside linkageslinking two deoxynucleosides are vulnerable to endonucleases and so incertain embodiments those linkages are phosphorothioate or othermodified linkage. Internucleoside linkages between a modified nucleosideand a deoxynucleoside where the deoxynucleoside is on the 5′ side of thelinkage deoxynucleosides are vulnerable to endonucleases and so incertain embodiments those linkages are phosphorothioate or othermodified linkage. Internucleoside linkages between two modifiednucleosides of certain types and between a deoxynucleoside and amodified nucleoside of certain type where the modified nucleoside is atthe 5′ side of the linkage are sufficiently resistant to nucleasedigestion, that the linkage can be phosphodiester.

In certain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 16 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 15 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 14 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 13 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 12 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 11 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 10 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 9 phosphorthioate linkages. Incertain embodiments, the antisense oligonucleotide of a conjugatedantisense compound comprises fewer than 8 phosphorthioate linkages.

In certain embodiments, antisense compounds comprising one or moreconjugate group described herein has increased activity and/or potencyand/or tolerability compared to a parent antisense compound lacking suchone or more conjugate group. Accordingly, in certain embodiments,attachment of such conjugate groups to an oligonucleotide is desirable.Such conjugate groups may be attached at the 5′-, and/or 3′-end of anoligonucleotide. In certain instances, attachment at the 5′-end issynthetically desirable. Typically, oligonucleotides are synthesized byattachment of the 3′ terminal nucleoside to a solid support andsequential coupling of nucleosides from 3′ to 5′ using techniques thatare well known in the art. Accordingly if a conjugate group is desiredat the 3′-terminus, one may (1) attach the conjugate group to the3′-terminal nucleoside and attach that conjugated nucleoside to thesolid support for subsequent preparation of the oligonucleotide or (2)attach the conjugate group to the 3′-terminal nucleoside of a completedoligonucleotide after synthesis. Neither of these approaches is veryefficient and thus both are costly. In particular, attachment of theconjugated nucleoside to the solid support, while demonstrated in theExamples herein, is an inefficient process. In certain embodiments,attaching a conjugate group to the 5′-terminal nucleoside issynthetically easier than attachment at the 3′-end. One may attach anon-conjugated 3′ terminal nucleoside to the solid support and preparethe oligonucleotide using standard and well characterized reactions. Onethen needs only to attach a 5′nucleoside having a conjugate group at thefinal coupling step. In certain embodiments, this is more efficient thanattaching a conjugated nucleoside directly to the solid support as istypically done to prepare a 3′-conjugated oligonucleotide. The Examplesherein demonstrate attachment at the 5′-end. In addition, certainconjugate groups have synthetic advantages. For Example, certainconjugate groups comprising phosphorus linkage groups are syntheticallysimpler and more efficiently prepared than other conjugate groups,including conjugate groups reported previously (e.g WO/2012/037254).

In certain embodiments, conjugated antisense compounds are administeredto a subject. In such embodiments, antisense compounds comprising one ormore conjugate group described herein has increased activity and/orpotency and/or tolerability compared to a parent antisense compoundlacking such one or more conjugate group. Without being bound bymechanism, it is believed that the conjugate group helps withdistribution, delivery, and/or uptake into a target cell or tissue. Incertain embodiments, once inside the target cell or tissue, it isdesirable that all or part of the conjugate group to be cleaved torelease the active oligonucleotide. In certain embodiments, it is notnecessary that the entire conjugate group be cleaved from theoligonucleotide. For example, in Example 20 a conjugated oligonucleotidewas administered to mice and a number of different chemical species,each comprising a different portion of the conjugate group remaining onthe oligonucleotide, were detected (Table 23a). This conjugatedantisense compound demonstrated good potency (Table 23). Thus, incertain embodiments, such metabolite profile of multiple partialcleavage of the conjugate group does not interfere withactivity/potency. Nevertheless, in certain embodiments it is desirablethat a prodrug (conjugated oligonucleotide) yield a single activecompound. In certain instances, if multiple forms of the active compoundare found, it may be necessary to determine relative amounts andactivities for each one. In certain embodiments where regulatory reviewis required (e.g., USFDA or counterpart) it is desirable to have asingle (or predominantly single) active species. In certain suchembodiments, it is desirable that such single active species be theantisense oligonucleotide lacking any portion of the conjugate group. Incertain embodiments, conjugate groups at the 5′-end are more likely toresult in complete metabolism of the conjugate group. Without beingbound by mechanism it may be that endogenous enzymes responsible formetabolism at the 5′ end (e.g., 5′ nucleases) are more active/efficientthan the 3′ counterparts. In certain embodiments, the specific conjugategroups are more amenable to metabolism to a single active species. Incertain embodiments, certain conjugate groups are more amenable tometabolism to the oligonucleotide.

D. Antisense

In certain embodiments, oligomeric compounds of the present inventionare antisense compounds. In such embodiments, the oligomeric compound iscomplementary to a target nucleic acid. In certain embodiments, a targetnucleic acid is an RNA. In certain embodiments, a target nucleic acid isa non-coding RNA. In certain embodiments, a target nucleic acid encodesa protein. In certain embodiments, a target nucleic acid is selectedfrom a mRNA, a pre-mRNA, a microRNA, a non-coding RNA, including smallnon-coding RNA, and a promoter-directed RNA. In certain embodiments,oligomeric compounds are at least partially complementary to more thanone target nucleic acid. For example, oligomeric compounds of thepresent invention may be microRNA mimics, which typically bind tomultiple targets.

In certain embodiments, antisense compounds comprise a portion having anucleobase sequence at least 70% complementary to the nucleobasesequence of a target nucleic acid. In certain embodiments, antisensecompounds comprise a portion having a nucleobase sequence at least 80%complementary to the nucleobase sequence of a target nucleic acid. Incertain embodiments, antisense compounds comprise a portion having anucleobase sequence at least 90% complementary to the nucleobasesequence of a target nucleic acid. In certain embodiments, antisensecompounds comprise a portion having a nucleobase sequence at least 95%complementary to the nucleobase sequence of a target nucleic acid. Incertain embodiments, antisense compounds comprise a portion having anucleobase sequence at least 98% complementary to the nucleobasesequence of a target nucleic acid. In certain embodiments, antisensecompounds comprise a portion having a nucleobase sequence that is 100%complementary to the nucleobase sequence of a target nucleic acid. Incertain embodiments, antisense compounds are at least 70%, 80%, 90%,95%, 98%, or 100% complementary to the nucleobase sequence of a targetnucleic acid over the entire length of the antisense compound.

Antisense mechanisms include any mechanism involving the hybridizationof an oligomeric compound with target nucleic acid, wherein thehybridization results in a biological effect. In certain embodiments,such hybridization results in either target nucleic acid degradation oroccupancy with concomitant inhibition or stimulation of the cellularmachinery involving, for example, translation, transcription, orpolyadenylation of the target nucleic acid or of a nucleic acid withwhich the target nucleic acid may otherwise interact.

One type of antisense mechanism involving degradation of target RNA isRNase H mediated antisense. RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. It is known in the art thatsingle-stranded antisense compounds which are “DNA-like” elicit RNase Hactivity in mammalian cells. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof DNA-like oligonucleotide-mediated inhibition of gene expression.

Antisense mechanisms also include, without limitation RNAi mechanisms,which utilize the RISC pathway. Such RNAi mechanisms include, withoutlimitation siRNA, ssRNA and microRNA mechanisms. Such mechanisms includecreation of a microRNA mimic and/or an anti-microRNA.

Antisense mechanisms also include, without limitation, mechanisms thathybridize or mimic non-coding RNA other than microRNA or mRNA. Suchnon-coding RNA includes, but is not limited to promoter-directed RNA andshort and long RNA that effects transcription or translation of one ormore nucleic acids.

In certain embodiments, oligonucleotides comprising conjugates describedherein are RNAi compounds. In certain embodiments, oligomericoligonucleotides comprising conjugates described herein are ssRNAcompounds. In certain embodiments, oligonucleotides comprisingconjugates described herein are paired with a second oligomeric compoundto form an siRNA. In certain such embodiments, the second oligomericcompound also comprises a conjugate. In certain embodiments, the secondoligomeric compound is any modified or unmodified nucleic acid. Incertain embodiments, the oligonucleotides comprising conjugatesdescribed herein is the antisense strand in an siRNA compound. Incertain embodiments, the oligonucleotides comprising conjugatesdescribed herein is the sense strand in an siRNA compound. Inembodiments in which the conjugated oligomeric compound isdouble-stranded siRnA, the conjugate may be on the sense strand, theantisense strand or both the sense strand and the antisense strand.

D. Apolipoprotein C-III (apoCIII)

In certain embodiments, conjugated antisense compounds target anyApoCIII nucleic acid. In certain embodiments, the target nucleic acidencodes an ApoCIII target protein that is clinically relevant. In suchembodiments, modulation of the target nucleic acid results in clinicalbenefit.

The targeting process usually includes determination of at least onetarget region, segment, or site within the target nucleic acid for theantisense interaction to occur such that the desired effect will result.

In certain embodiments, a target region is a structurally defined regionof the nucleic acid. For example, in certain such embodiments, a targetregion may encompass a 3′ UTR, a 5′ UTR, an exon, an intron, a codingregion, a translation initiation region, translation termination region,or other defined nucleic acid region or target segment.

In certain embodiments, a target segment is at least about an8-nucleobase portion of a target region to which a conjugated antisensecompound is targeted. Target segments can include DNA or RNA sequencesthat comprise at least 8 consecutive nucleobases from the 5′-terminus ofone of the target segments (the remaining nucleobases being aconsecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA comprises about 8 to about 30 nucleobases). Targetsegments are also represented by DNA or RNA sequences that comprise atleast 8 consecutive nucleobases from the 3′-terminus of one of thetarget segments (the remaining nucleobases being a consecutive stretchof the same DNA or RNA beginning immediately downstream of the3′-terminus of the target segment and continuing until the DNA or RNAcomprises about 8 to about 30 nucleobases). Target segments can also berepresented by DNA or RNA sequences that comprise at least 8 consecutivenucleobases from an internal portion of the sequence of a targetsegment, and may extend in either or both directions until theconjugated antisense compound comprises about 8 to about 30 nucleobases.

In certain embodiments, antisense compounds targeted to an ApoCIIInucleic acid can be modified as described herein. In certainembodiments, the antisense compounds can have a modified sugar moiety,an unmodified sugar moiety or a mixture of modified and unmodified sugarmoieties as described herein. In certain embodiments, the antisensecompounds can have a modified internucleoside linkage, an unmodifiedinternucleoside linkage or a mixture of modified and unmodifiedinternucleoside linkages as described herein. In certain embodiments,the antisense compounds can have a modified nucleobase, an unmodifiednucleobase or a mixture of modified and unmodified nucleobases asdescribed herein. In certain embodiments, the antisense compounds canhave a motif as described herein.

In certain embodiments, antisense compounds targeted to ApoCIII nucleicacids can be conjugated as described herein.

ApoCIII is a constituent of HDL and of triglyceride (TG)-richlipoproteins. Elevated ApoCIII levels are associated with elevated TGlevels and diseases such as cardiovascular disease, metabolic syndrome,obesity and diabetes. Elevated TG levels are associated withpancreatitis. ApoCIII slows clearance of TG-rich lipoproteins byinhibiting lipolysis through inhibition of lipoprotein lipase (LPL) andthrough interfering with lipoprotein binding to cell-surfaceglycosaminoglycan matrix. Antisense compounds targeting ApoCIII havebeen previously disclosed in WO2004/093783 and WO2012/149495, eachherein incorporated by reference in its entirety.

Certain Conjugated Antisense Compounds Targeted to an ApoCIII NucleicAcid

In certain embodiments, conjugated antisense compounds are targeted toan ApoCIII nucleic acid having the sequence of any of GENBANK® AccessionNo. NM_000040.1 (incorporated herein as SEQ ID NO: 1); GENBANK AccessionNo. NT_033899.8 truncated from nucleotides 20262640 to 20266603(incorporated herein as SEQ ID NO: 2); and GenBank Accession No.NT_035088.1 truncated from nucleotides 6238608 to U.S. Pat. No.6,242,565 (incorporated herein as SEQ ID NO: 3). In certain suchembodiments, a conjugated antisense compound is at least 90%, at least95%, or 100% complementary to any of SEQ ID NOs: 1-3.

In certain embodiments, a conjugated antisense compound targeted to SEQID NO: 1 comprises an at least 8 consecutive nucleobase sequence of SEQID NO: 87. In certain embodiments, a conjugated antisense compoundtargeted to SEQ ID NO: 1 comprises a nucleobase sequence of SEQ ID NO:87.

TABLE A Antisense Compounds targeted to ApoCIII SEQ ID NO: 1 TargetStart SEQ ID ISIS No Site Sequence (5′-3′) Motif NO 304801 508AGCTTCTTGTCCAGCTTTAT eeeeeddddddddddeeeee 87 647535 508AGCTTCTTGTCCAGCTTTAT eeeeeddddddddddeeeeeod 87 616468 508AGCTTCTTGTCCAGCTTTAT eeeeeddddddddddeeeee 87 647536 508AGCTTCTTGTCCAGCTTTAT eeoeoeoeoddddddddddeoe 87 oeeeod

ApoCIII Therapeutic Indications

In certain embodiments, the invention provides methods for using aconjugated antisense compound targeted to an ApoCIII nucleic acid formodulating the expression of ApoCIII in a subject. In certainembodiments, the expression of ApoCIII is reduced.

In certain embodiments, the invention provides methods for using aconjugated antisense compound targeted to an ApoCIII nucleic acid in apharmaceutical composition for treating a subject. In certainembodiments, the subject has a cardiovascular and/or metabolic disease,disorder or condition. In certain embodiments, the subject hashypertriglyceridemia, non-familial hypertriglyceridemia, familialhypertriglyceridemia, heterozygous familial hypertriglyceridemia,homozygous familial hypertriglyceridemia, mixed dyslipidemia,atherosclerosis, a risk of developing atherosclerosis, coronary heartdisease, a history of coronary heart disease, early onset coronary heartdisease, one or more risk factors for coronary heart disease, type IIdiabetes, type II diabetes with dyslipidemia, dyslipidemia,hyperlipidemia, hypercholesterolemia, hyperfattyacidemia, hepaticsteatosis, non-alcoholic steatohepatitis, pancreatitis and/ornon-alcoholic fatty liver disease.

In certain embodiments, the invention provides methods for using aconjugated antisense compound targeted to an ApoCIII nucleic acid in thepreparation of a medicament.

E. Certain Nucleic Acid GalNAc Conjugates

In certain embodiments, conjugated antisense compounds compriseantisense compounds having the nucleobase sequence and modifications ofthe antisense compounds in the Table below attached to a GalNAcconjugate. All internucleoside linkages are phosphorothioateinternucleoside linkages unless otherwise indicated. A subscript “1”indicates an LNA bicyclic nucleoside. A subscript “d” indicates a2′-deoxy nucleoside. A subscript “e” indicates a 2′-MOE modifiednucleoside. A “V” indicates a 2-amino-2′-deoxyadenosine.

TABLE B SEQ Sequence Internucleoside ID 5′ to 3′ Motif ChemistryLinkages NO.T_(l)G_(l)G_(l)C_(d)A_(d)A_(d)G_(d)C_(d)A_(d)T_(d)C_(d)C_(d)T_(l)G_(l)T_(l)A_(d)3-9-3-1 LNA/deoxy phosphorothioate 222C_(l)T_(l)C_(l)A_(l)A_(d)T_(d)C_(d)C_(d)A_(d)T_(d)G_(d)G_(d)C_(l)A_(l)G_(l)C_(d)4-8-3-1 LNA/deoxy phosphorothioate 223A_(l)C_(l)C_(l)A_(d)A_(d)G_(d)T_(d)T_(d)T_(d)C_(d)T_(d)T_(d)C_(d)A_(l)G_(l)C_(l)3-10-3 LNA/deoxy phosphorothioate 224G_(l)C_(l)A_(d)T_(d)T_(d)G_(d)G_(d)T_(d)A_(d)T_(d)T_(l)C_(l)A_(l) 2-8-3LNA/deoxy phosphorothioate 225T_(l)T_(l)C_(l)A_(l)G_(l)C_(d)A_(d)T_(d)T_(d)G_(d)G_(d)T_(d)A_(d)T_(d)T_(d)C_(l)A_(l)G_(l)T_(l)G_(l)5-10-5 LNA/deoxy phosphorothioate 226C_(l)A_(l)G_(l)C_(d)A_(d)T_(d)T_(d)G_(d)G_(d)T_(d)A_(d)T_(d)T_(l)C_(l)A_(l)G_(d)3-10-3 LNA/deoxy phosphorothioate 227C_(l)A_(l)G_(l)C_(d)A_(d)T_(d)T_(d)G_(d)G_(d)T_(d)A_(d)T_(d)T_(l)C_(l)A_(l)3-9-3 LNA/deoxy phosphorothioate 228A_(l)G_(l)C_(l)A_(d)T_(d)T_(d)G_(d)G_(d)T_(d)A_(d)T_(d)T_(l)C_(l)A_(l)3-8-3 LNA/deoxy phosphorothioate 229G_(l)C_(l)A_(d)T_(d)T_(d)G_(d)G_(d)T_(d)A_(d)T_(d)T_(l)C_(l) 2-8-2LNA/deoxy phosphorothioate 230 CGGCATGTCTATTTTGTA phosphorothioate 231GGCTAAATCGCTCCACCAAG phosphorothioate 232 CTCTAGCGTCTTAAAGCCGAphosphorothioate 233 GCTGCATGATCTCCTTGGCG phosphorothioate 234ACGTTGAGGGGCATCGTCGC Morpholino 235 GGGTCTGCVGCGGGVTGGT phosphorothioate236 GTTVCTVCTTCCVCCTGCCTG phosphorothioate 237 TATCCGGAGGGCTCGCCATGCTGCTphosphorothioate 238T_(e)C_(e)C_(e)C_(e)G_(e)C_(e)CTGTGACAT_(e)G_(e)C_(e)A_(e)T_(e)T_(e)6-8-6 MOE/deoxy 239C_(e)A_(e)G_(e)C_(e)AGCAGAGTCTTCAT_(e)C_(e)A_(e)T_(e) 4-13-4 MOE/deoxy240G_(e)G_(e)G_(e)A_(e)C_(d)G_(d)C_(d)G_(d)G_(d)C_(d)G_(d)C_(d)T_(d)C_(d)G_(d)G_(d)T_(e)C_(e)A_(e)T_(e)4-12-4 MOE/deoxy 241C_(e)C_(e)A_(e)C_(e)A_(e)A_(d)G_(d)C_(d)T_(d)G_(d)T_(d)C_(d)C_(d)A_(d)G_(d)T_(e)C_(e)T_(e)A_(e)A_(e)5-10-5 MOE/deoxy 242C_(e)C_(e)G_(e)C_(d)A_(d)G_(d)C_(d)C_(d)A_(d)T_(d)G_(d)C_(d)G_(e)C_(e)T_(e)C_(e)T_(e)T_(e)G_(e)G_(e)3-9-8 MOE/deoxy 243

F. Certain Pharmaceutical Compositions

In certain embodiments, the present disclosure provides pharmaceuticalcompositions comprising one or more antisense compound. In certainembodiments, such pharmaceutical composition comprises a suitablepharmaceutically acceptable diluent or carrier. In certain embodiments,a pharmaceutical composition comprises a sterile saline solution and oneor more antisense compound. In certain embodiments, such pharmaceuticalcomposition consists of a sterile saline solution and one or moreantisense compound. In certain embodiments, the sterile saline ispharmaceutical grade saline. In certain embodiments, a pharmaceuticalcomposition comprises one or more antisense compound and sterile water.In certain embodiments, a pharmaceutical composition consists of one ormore antisense compound and sterile water. In certain embodiments, thesterile saline is pharmaceutical grade water. In certain embodiments, apharmaceutical composition comprises one or more antisense compound andphosphate-buffered saline (PBS). In certain embodiments, apharmaceutical composition consists of one or more antisense compoundand sterile phosphate-buffered saline (PBS). In certain embodiments, thesterile saline is pharmaceutical grade PBS.

In certain embodiments, antisense compounds may be admixed withpharmaceutically acceptable active and/or inert substances for thepreparation of pharmaceutical compositions or formulations. Compositionsand methods for the formulation of pharmaceutical compositions depend ona number of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters. Incertain embodiments, pharmaceutical compositions comprising antisensecompounds comprise one or more oligonucleotide which, uponadministration to an animal, including a human, is capable of providing(directly or indirectly) the biologically active metabolite or residuethereof. Accordingly, for example, the disclosure is also drawn topharmaceutically acceptable salts of antisense compounds, prodrugs,pharmaceutically acceptable salts of such prodrugs, and otherbioequivalents. Suitable pharmaceutically acceptable salts include, butare not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an oligonucleotide which are cleaved by endogenousnucleases within the body, to form the active antisense oligonucleotide.

Lipid moieties have been used in nucleic acid therapies in a variety ofmethods. In certain such methods, the nucleic acid is introduced intopreformed liposomes or lipoplexes made of mixtures of cationic lipidsand neutral lipids. In certain methods, DNA complexes with mono- orpoly-cationic lipids are formed without the presence of a neutral lipid.In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to a particular cell or tissue.In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to fat tissue. In certainembodiments, a lipid moiety is selected to increase distribution of apharmaceutical agent to muscle tissue.

In certain embodiments, pharmaceutical compositions provided hereincomprise one or more modified oligonucleotides and one or moreexcipients. In certain such embodiments, excipients are selected fromwater, salt solutions, alcohol, polyethylene glycols, gelatin, lactose,amylase, magnesium stearate, talc, silicic acid, viscous paraffin,hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical composition provided hereincomprises a delivery system. Examples of delivery systems include, butare not limited to, liposomes and emulsions. Certain delivery systemsare useful for preparing certain pharmaceutical compositions includingthose comprising hydrophobic compounds. In certain embodiments, certainorganic solvents such as dimethylsulfoxide are used.

In certain embodiments, a pharmaceutical composition provided hereincomprises one or more tissue-specific delivery molecules designed todeliver the one or more pharmaceutical agents of the present disclosureto specific tissues or cell types. For example, in certain embodiments,pharmaceutical compositions include liposomes coated with atissue-specific antibody.

In certain embodiments, a pharmaceutical composition provided hereincomprises a co-solvent system. Certain of such co-solvent systemscomprise, for example, benzyl alcohol, a nonpolar surfactant, awater-miscible organic polymer, and an aqueous phase. In certainembodiments, such co-solvent systems are used for hydrophobic compounds.A non-limiting example of such a co-solvent system is the VPD co-solventsystem, which is a solution of absolute ethanol comprising 3% w/v benzylalcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/vpolyethylene glycol 300. The proportions of such co-solvent systems maybe varied considerably without significantly altering their solubilityand toxicity characteristics. Furthermore, the identity of co-solventcomponents may be varied: for example, other surfactants may be usedinstead of Polysorbate 80™; the fraction size of polyethylene glycol maybe varied; other biocompatible polymers may replace polyethylene glycol,e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides maysubstitute for dextrose.

In certain embodiments, a pharmaceutical composition provided herein isprepared for oral administration. In certain embodiments, pharmaceuticalcompositions are prepared for buccal administration.

In certain embodiments, a pharmaceutical composition is prepared foradministration by injection (e.g., intravenous, subcutaneous,intramuscular, etc.). In certain of such embodiments, a pharmaceuticalcomposition comprises a carrier and is formulated in aqueous solution,such as water or physiologically compatible buffers such as Hanks'ssolution, Ringer's solution, or physiological saline buffer. In certainembodiments, other ingredients are included (e.g., ingredients that aidin solubility or serve as preservatives). In certain embodiments,injectable suspensions are prepared using appropriate liquid carriers,suspending agents and the like. Certain pharmaceutical compositions forinjection are presented in unit dosage form, e.g., in ampoules or inmulti-dose containers. Certain pharmaceutical compositions for injectionare suspensions, solutions or emulsions in oily or aqueous vehicles, andmay contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Certain solvents suitable for use in pharmaceuticalcompositions for injection include, but are not limited to, lipophilicsolvents and fatty oils, such as sesame oil, synthetic fatty acidesters, such as ethyl oleate or triglycerides, and liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, such suspensions may also contain suitablestabilizers or agents that increase the solubility of the pharmaceuticalagents to allow for the preparation of highly concentrated solutions.

In certain embodiments, a pharmaceutical composition is prepared fortransmucosal administration. In certain of such embodiments penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

In certain embodiments, a pharmaceutical composition provided hereincomprises an oligonucleotide in a therapeutically effective amount. Incertain embodiments, the therapeutically effective amount is sufficientto prevent, alleviate or ameliorate symptoms of a disease or to prolongthe survival of the subject being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art.

In certain embodiments, one or more modified oligonucleotide providedherein is formulated as a prodrug. In certain embodiments, upon in vivoadministration, a prodrug is chemically converted to the biologically,pharmaceutically or therapeutically more active form of anoligonucleotide. In certain embodiments, prodrugs are useful becausethey are easier to administer than the corresponding active form. Forexample, in certain instances, a prodrug may be more bioavailable (e.g.,through oral administration) than is the corresponding active form. Incertain instances, a prodrug may have improved solubility compared tothe corresponding active form. In certain embodiments, prodrugs are lesswater soluble than the corresponding active form. In certain instances,such prodrugs possess superior transmittal across cell membranes, wherewater solubility is detrimental to mobility. In certain embodiments, aprodrug is an ester. In certain such embodiments, the ester ismetabolically hydrolyzed to carboxylic acid upon administration. Incertain instances the carboxylic acid containing compound is thecorresponding active form. In certain embodiments, a prodrug comprises ashort peptide (polyaminoacid) bound to an acid group. In certain of suchembodiments, the peptide is cleaved upon administration to form thecorresponding active form.

In certain embodiments, the present disclosure provides compositions andmethods for reducing the amount or activity of a target nucleic acid ina cell. In certain embodiments, the cell is in an animal. In certainembodiments, the animal is a mammal. In certain embodiments, the animalis a rodent. In certain embodiments, the animal is a primate. In certainembodiments, the animal is a non-human primate. In certain embodiments,the animal is a human.

In certain embodiments, the present disclosure provides methods ofadministering a pharmaceutical composition comprising an oligonucleotideof the present disclosure to an animal. Suitable administration routesinclude, but are not limited to, oral, rectal, transmucosal, intestinal,enteral, topical, suppository, through inhalation, intrathecal,intracerebroventricular, intraperitoneal, intranasal, intraocular,intratumoral, and parenteral (e.g., intravenous, intramuscular,intramedullary, and subcutaneous). In certain embodiments,pharmaceutical intrathecals are administered to achieve local ratherthan systemic exposures. For example, pharmaceutical compositions may beinjected directly in the area of desired effect (e.g., into the liver).

Nonlimiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the references,GenBank accession numbers, and the like recited in the presentapplication is incorporated herein by reference in its entirety.

Although the sequence listing accompanying this filing identifies eachsequence as either “RNA” or “DNA” as required, in reality, thosesequences may be modified with any combination of chemicalmodifications. One of skill in the art will readily appreciate that suchdesignation as “RNA” or “DNA” to describe modified oligonucleotides is,in certain instances, arbitrary. For example, an oligonucleotidecomprising a nucleoside comprising a 2′-OH sugar moiety and a thyminebase could be described as a DNA having a modified sugar (2′-OH for thenatural 2′-H of DNA) or as an RNA having a modified base (thymine(methylated uracil) for natural uracil of RNA).

Accordingly, nucleic acid sequences provided herein, including, but notlimited to those in the sequence listing, are intended to encompassnucleic acids containing any combination of natural or modified RNAand/or DNA, including, but not limited to such nucleic acids havingmodified nucleobases. By way of further example and without limitation,an oligonucleotide having the nucleobase sequence “ATCGATCG” encompassesany oligonucleotides having such nucleobase sequence, whether modifiedor unmodified, including, but not limited to, such compounds comprisingRNA bases, such as those having sequence “AUCGAUCG” and those havingsome DNA bases and some RNA bases such as “AUCGATCG” andoligonucleotides having other modified bases, such as “AT^(me)CGAUCG,”wherein ^(me)C indicates a cytosine base comprising a methyl group atthe 5-position.

EXAMPLES

The following examples illustrate certain embodiments of the presentdisclosure and are not limiting. Moreover, where specific embodimentsare provided, the inventors have contemplated generic application ofthose specific embodiments. For example, disclosure of anoligonucleotide having a particular motif provides reasonable supportfor additional oligonucleotides having the same or similar motif. And,for example, where a particular high-affinity modification appears at aparticular position, other high-affinity modifications at the sameposition are considered suitable, unless otherwise indicated.

Example 1: General Method for the Preparation of Phosphoramidites,Compounds 1, 1a and 2

Bx is a heterocyclic base;

Compounds 1, 1a and 2 were prepared as per the procedures well known inthe art as described in the specification herein (see Seth et al.,Bioorg. Med. Chem., 2011, 21(4), 1122-1125, J. Org. Chem., 2010, 75(5),1569-1581, Nucleic Acids Symposium Series, 2008, 52(1), 553-554); andalso see published PCT International Applications (WO 2011/115818, WO2010/077578, WO2010/036698, WO2009/143369, WO 2009/006478, and WO2007/090071), and U.S. Pat. No. 7,569,686).

Example 2: Preparation of Compound 7

Compounds 3(2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-β-Dgalactopyranose orgalactosamine pentaacetate) is commercially available. Compound 5 wasprepared according to published procedures (Weber et al., J. Med. Chem.,1991, 34, 2692).

Example 3: Preparation of Compound 11

Compounds 8 and 9 are commercially available.

Example 4: Preparation of Compound 18

Compound 11 was prepared as per the procedures illustrated in Example 3.Compound 14 is commercially available. Compound 17 was prepared usingsimilar procedures reported by Rensen et al., J. Med. Chem., 2004, 47,5798-5808.

Example 5: Preparation of Compound 23

Compounds 19 and 21 are commercially available.

Example 6: Preparation of Compound 24

Compounds 18 and 23 were prepared as per the procedures illustrated inExamples 4 and 5.

Example 7: Preparation of Compound 25

Compound 24 was prepared as per the procedures illustrated in Example 6.

Example 8: Preparation of Compound 26

Compound 24 is prepared as per the procedures illustrated in Example 6.

Example 9: General Preparation of Conjugated ASOs Comprising GalNAc3-1at the 3′ Terminus, Compound 29

-   -   Wherein the protected GalNAc3-1 has the structure:

The GalNAc3 cluster portion of the conjugate group GalNAc3-1(GalNAc3-1_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. Wherein GalNAc3-1_(a) has the formula:

The solid support bound protected GalNAc3-1, Compound 25, was preparedas per the procedures illustrated in Example 7. Oligomeric Compound 29comprising GalNAc3-1 at the 3′ terminus was prepared using standardprocedures in automated DNA/RNA synthesis (see Dupouy et al., Angew.Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite building blocks,Compounds 1 and 1a were prepared as per the procedures illustrated inExample 1. The phosphoramidites illustrated are meant to berepresentative and not intended to be limiting as other phosphoramiditebuilding blocks can be used to prepare oligomeric compounds having apredetermined sequence and composition. The order and quantity ofphosphoramidites added to the solid support can be adjusted to preparegapped oligomeric compounds as described herein. Such gapped oligomericcompounds can have predetermined composition and base sequence asdictated by any given target.

Example 10: General Preparation Conjugated ASOs Comprising GalNAc3-1 atthe 5′ Terminus, Compound 34

The Unylinker™ 30 is commercially available. Oligomeric Compound 34comprising a GalNAc3-1 cluster at the 5′ terminus is prepared usingstandard procedures in automated DNA/RNA synthesis (see Dupouy et al.,Angew. Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite buildingblocks, Compounds 1 and 1a were prepared as per the proceduresillustrated in Example 1. The phosphoramidites illustrated are meant tobe representative and not intended to be limiting as otherphosphoramidite building blocks can be used to prepare an oligomericcompound having a predetermined sequence and composition. The order andquantity of phosphoramidites added to the solid support can be adjustedto prepare gapped oligomeric compounds as described herein. Such gappedoligomeric compounds can have predetermined composition and basesequence as dictated by any given target.

Example 11: Preparation of Compound 39

Compounds 4, 13 and 23 were prepared as per the procedures illustratedin Examples 2, 4, and 5. Compound 35 is prepared using similarprocedures published in Rouchaud et al., Eur. J. Org. Chem., 2011, 12,2346-2353.

Example 12: Preparation of Compound 40

Compound 38 is prepared as per the procedures illustrated in Example 11.

Example 13: Preparation of Compound 44

Compounds 23 and 36 are prepared as per the procedures illustrated inExamples 5 and 11. Compound 41 is prepared using similar procedurespublished in WO 2009082607.

Example 14: Preparation of Compound 45

Compound 43 is prepared as per the procedures illustrated in Example 13.

Example 15: Preparation of Compound 47

Compound 46 is commercially available.

Example 16: Preparation of Compound 53

Compounds 48 and 49 are commercially available. Compounds 17 and 47 areprepared as per the procedures illustrated in Examples 4 and 15.

Example 17: Preparation of Compound 54

Compound 53 is prepared as per the procedures illustrated in Example 16.

Example 18: Preparation of Compound 55

Compound 53 is prepared as per the procedures illustrated in Example 16.

Example 19: General Method for the Preparation of Conjugated ASOsComprising GalNAc3-1 at the 3′ Position Via Solid Phase Techniques(Preparation of ISIS 647535, 647536 and 651900)

Unless otherwise stated, all reagents and solutions used for thesynthesis of oligomeric compounds are purchased from commercial sources.Standard phosphoramidite building blocks and solid support are used forincorporation nucleoside residues which include for example T, A, G, and^(m)C residues. A 0.1 M solution of phosphoramidite in anhydrousacetonitrile was used for β-D-2′-deoxyribonucleoside and 2′-MOE.

The ASO syntheses were performed on ABI 394 synthesizer (1-2 μmol scale)or on GE Healthcare Bioscience ÄKTA oligopilot synthesizer (40-200 μmolscale) by the phosphoramidite coupling method on an GalNAc3-1 loadedVIMAD solid support (110 μmol/g, Guzaev et al., 2003) packed in thecolumn. For the coupling step, the phosphoramidites were delivered 4fold excess over the loading on the solid support and phosphoramiditecondensation was carried out for 10 min. All other steps followedstandard protocols supplied by the manufacturer. A solution of 6%dichloroacetic acid in toluene was used for removing dimethoxytrityl(DMT) group from 5′-hydroxyl group of the nucleotide.4,5-Dicyanoimidazole (0.7 M) in anhydrous CH₃CN was used as activatorduring coupling step. Phosphorothioate linkages were introduced bysulfurization with 0.1 M solution of xanthane hydride in 1:1pyridine/CH₃CN for a contact time of 3 minutes. A solution of 20%tert-butylhydroperoxide in CH₃CN containing 6% water was used as anoxidizing agent to provide phosphodiester internucleoside linkages witha contact time of 12 minutes.

After the desired sequence was assembled, the cyanoethyl phosphateprotecting groups were deprotected using a 1:1 (v/v) mixture oftriethylamine and acetonitrile with a contact time of 45 minutes. Thesolid-support bound ASOs were suspended in aqueous ammonia (28-30 wt %)and heated at 55° C. for 6 h.

The unbound ASOs were then filtered and the ammonia was boiled off. Theresidue was purified by high pressure liquid chromatography on a stronganion exchange column (GE Healthcare Bioscience, Source 30Q, 30 μm,2.54×8 cm, A=100 mM ammonium acetate in 30% aqueous CH₃CN, B=1.5 M NaBrin A, 0-40% of B in 60 min, flow 14 mL min-1, =260 nm). The residue wasdesalted by HPLC on a reverse phase column to yield the desired ASOs inan isolated yield of 15-30% based on the initial loading on the solidsupport. The ASOs were characterized by ion-pair-HPLC coupled MSanalysis with Agilent 1100 MSD system.

Antisense oligonucleotides not comprising a conjugate were synthesizedusing standard oligonucleotide synthesis procedures well known in theart.

Using these methods, three separate antisense compounds targeting ApoCIII were prepared. As summarized in Table 17, below, each of the threeantisense compounds targeting ApoC III had the same nucleobase sequence;ISIS 304801 is a 5-10-5 MOE gapmer having all phosphorothioate linkages;ISIS 647535 is the same as ISIS 304801, except that it had a GalNAc₃-1conjugated at its 3′end; and ISIS 647536 is the same as ISIS 647535except that certain internucleoside linkages of that compound arephosphodiester linkages. As further summarized in Table 17, two separateantisense compounds targeting SRB-1 were synthesized. ISIS 440762 was a2-10-2 cEt gapmer with all phosphorothioate internucleoside linkages;ISIS 651900 is the same as ISIS 440762, except that it included aGalNAc₃-1 at its 3′-end.

TABLE 17 Modified ASO targeting ApoC III and SRB-1 SEQ CalCd Observed IDASO Sequence (5′ to 3′) Target Mass Mass No. ISIS A_(es)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds) T_(es)T_(es)T_(es)A_(es)T_(e) ApoC7165.4 7164.4 244 304801 III ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(eo) A _(do′) - ApoC 9239.5 9237.8245 647535 GalNAc ₃ -1 _(a) III ISIS A_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(eo) A _(do′) - ApoC 9142.9 9140.8245 647536 GalNAc ₃ -1 _(a) III ISIS T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k)SRB-1 4647.0 4646.4 246 440762 ISIS T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ko) A_(do′) -GalNAc ₃ -1 _(a) SRB-1 6721.1 6719.4 247 651900Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicatesβ-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside(e.g. cEt); “s” indicates phosphorothioate internucleoside linkages(PS); “o” indicates phosphodiester internucleoside linkages (PO); and“o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates5-methylcytosines. “GalNAc₃-1” indicates a conjugate group having thestructure shown previously in Example 9. Note that GalNAc₃-1 comprises acleavable adenosine which links the ASO to remainder of the conjugate,which is designated “GalNAc₃-1_(a).” This nomenclature is used in theabove table to show the full nucleobase sequence, including theadenosine, which is part of the conjugate. Thus, in the above table, thesequences could also be listed as ending with “GalNAc₃-1” with the“A_(do)” omitted. This convention of using the subscript “a” to indicatethe portion of a conjugate group lacking a cleavable nucleoside orcleavable moiety is used throughout these Examples. This portion of aconjugate group lacking the cleavable moiety is referred to herein as a“cluster” or “conjugate cluster” or “GalNAc3 cluster.” In certaininstances it is convenient to describe a conjugate group by separatelyproviding its cluster and its cleavable moiety.

Example 20: Dose-Dependent Antisense Inhibition of Human ApoC III inhuApoC III Transgenic Mice

ISIS 304801 and ISIS 647535, each targeting human ApoC III and describedabove, were separately tested and evaluated in a dose-dependent studyfor their ability to inhibit human ApoC III in human ApoC III transgenicmice.

Treatment

Human ApoCIII transgenic mice were maintained on a 12-hour light/darkcycle and fed ad libitum Teklad lab chow. Animals were acclimated for atleast 7 days in the research facility before initiation of theexperiment. ASOs were prepared in PBS and sterilized by filteringthrough a 0.2 micron filter. ASOs were dissolved in 0.9% PBS forinjection.

Human ApoC III transgenic mice were injected intraperitoneally once aweek for two weeks with ISIS 304801 or 647535 at 0.08, 0.25. 0.75, 2.25or 6.75 μmol/kg or with PBS as a control. Each treatment group consistedof 4 animals. Forty-eight hours after the administration of the lastdose, blood was drawn from each mouse and the mice were sacrificed andtissues were collected.

ApoC III mRNA Analysis

ApoC III mRNA levels in the mice's livers were determined usingreal-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. ApoC IIImRNA levels were determined relative to total RNA (using Ribogreen),prior to normalization to PBS-treated control. The results below arepresented as the average percent of ApoC III mRNA levels for eachtreatment group, normalized to PBS-treated control and are denoted as “%PBS”. The half maximal effective dosage (ED₅₀) of each ASO is alsopresented in Table 18, below.

As illustrated, both antisense compounds reduced ApoC III RNA relativeto the PBS control. Further, the antisense compound conjugated toGalNAc₃-1 (ISIS 647535) was substantially more potent than the antisensecompound lacking the GalNAc₃-1 conjugate (ISIS 304801).

TABLE 18 Effect of ASO treatment on ApoC III mRNA levels in human ApoCIII transgenic mice Inter- nucleoside SEQ Dose % ED₅₀ 3′ linkage/ ID ASO(μmol/kg) PBS (μmol/kg) Conjugate Length No. PBS 0 100 — — — ISIS 0.0895 0.77 None PS/20 244 304801 0.75 42 2.25 32 6.75 19 ISIS 0.08 50 0.074GalNAc₃-1 PS/20 245 647535 0.75 15 2.25 17 6.75 8

ApoC III Protein Analysis (Turbidometric Assay)

Plasma ApoC III protein analysis was determined using proceduresreported by Graham et al, Circulation Research, published online beforeprint Mar. 29, 2013.

Approximately 100 μl of plasma isolated from mice was analyzed withoutdilution using an Olympus Clinical Analyzer and a commercially availableturbidometric ApoC III assay (Kamiya, Cat# KAI-006, Kamiya Biomedical,Seattle, Wash.). The assay protocol was performed as described by thevendor.

As shown in the Table 19 below, both antisense compounds reduced ApoCIII protein relative to the PBS control. Further, the antisense compoundconjugated to GalNAc₃-1 (ISIS 647535) was substantially more potent thanthe antisense compound lacking the GalNAc₃-1 conjugate (ISIS 304801).

TABLE 19 Effect of ASO treatment on ApoC III plasma protein levels inhuman ApoC III transgenic mice Inter- nucleoside SEQ Dose % ED₅₀ 3′Linkage/ ID ASO (μmol/kg) PBS (μmol/kg) Conjugate Length No. PBS 0 100 —— — ISIS 0.08 86 0.73 None PS/20 244 304801 0.75 51 2.25 23 6.75 13 ISIS0.08 72 0.19 Ga1NAc₃-1 PS/20 245 647535 0.75 14 2.25 12 6.75 11

Plasma triglycerides and cholesterol were extracted by the method ofBligh and Dyer (Bligh, E. G. and Dyer, W. J. Can. J. Biochem. Physiol.37: 911-917, 1959)(Bligh, E and Dyer, W, Can J Biochem Physiol, 37,911-917, 1959)(Bligh, E and Dyer, W, Can J Biochem Physiol, 37, 911-917,1959) and measured by using a Beckmann Coulter clinical analyzer andcommercially available reagents.

The triglyceride levels were measured relative to PBS injected mice andare denoted as “% PBS”. Results are presented in Table 20. Asillustrated, both antisense compounds lowered triglyceride levels.Further, the antisense compound conjugated to GalNAc₃-1 (ISIS 647535)was substantially more potent than the antisense compound lacking theGalNAc₃-1 conjugate (ISIS 304801).

TABLE 20 Effect of ASO treatment on triglyceride levels in transgenicmice Inter- nucleoside SEQ Dose 0% ED₅₀ 3′ Linkage/ ID ASO (μmol/kg) PBS(μmol/kg) Conjugate Length No. PBS 0 100 — — — ISIS 0.08 87 0.63 NonePS/20 244 304801 0.75 46 2.25 21 6.75 12 ISIS 0.08 65 0.13 GalNAc₃-1PS/20 245 647535 0.75 9 2.25 8 6.75 9

Plasma samples were analyzed by HPLC to determine the amount of totalcholesterol and of different fractions of cholesterol (HDL and LDL).Results are presented in Tables 21 and 22. As illustrated, bothantisense compounds lowered total cholesterol levels; both lowered LDL;and both raised HDL. Further, the antisense compound conjugated toGalNAc₃-1 (ISIS 647535) was substantially more potent than the antisensecompound lacking the GalNAc₃-1 conjugate (ISIS 304801). An increase inHDL and a decrease in LDL levels is a cardiovascular beneficial effectof antisense inhibition of ApoC III.

TABLE 21 Effect of ASO treatment on total cholesterol levels intransgenic mice Inter- Total nucleoside SEQ Dose Cholesterol 3′ Linkage/ID ASO (μmol/kg) (mg/dL) Conjugate Length No. PBS 0 257 — — ISIS 0.08226 None PS/20 244 304801 0.75 164 2.25 110 6.75 82 ISIS 0.08 230GalNAc₃-1 PS/20 245 647535 0.75 82 2.25 86 6.75 99

TABLE 22 Effect of ASO treatment on HDL and LDL cholesterol levels intransgenic mice Inter- nucleoside SEQ Dose HDL LDL 3′ Linkage/ ID ASO(μmol/kg) (mg/dL) (mg/dL) Conjugate Length No. PBS 0 17 28 — — ISIS 0.0817 23 None PS/20 244 304801 0.75 27 12 2.25 50 4 6.75 45 2 ISIS 0.08 2121 GalNAc₃-1 PS/20 245 647535 0.75 44 2 2.25 50 2 6.75 58 2

Pharmacokinetics Analysis (PK)

The PK of the ASOs was also evaluated. Liver and kidney samples wereminced and extracted using standard protocols. Samples were analyzed onMSD1 utilizing IP-HPLC-MS. The tissue level (μg/g) of full-length ISIS304801 and 647535 was measured and the results are provided in Table 23.As illustrated, liver concentrations of total full-length antisensecompounds were similar for the two antisense compounds. Thus, eventhough the GalNAc₃-1-conjugated antisense compound is more active in theliver (as demonstrated by the RNA and protein data above), it is notpresent at substantially higher concentration in the liver. Indeed, thecalculated EC₅₀ (provided in Table 23) confirms that the observedincrease in potency of the conjugated compound cannot be entirelyattributed to increased accumulation. This result suggests that theconjugate improved potency by a mechanism other than liver accumulationalone, possibly by improving the productive uptake of the antisensecompound into cells.

The results also show that the concentration of GalNAc₃-1 conjugatedantisense compound in the kidney is lower than that of antisensecompound lacking the GalNAc conjugate. This has several beneficialtherapeutic implications. For therapeutic indications where activity inthe kidney is not sought, exposure to kidney risks kidney toxicitywithout corresponding benefit. Moreover, high concentration in kidneytypically results in loss of compound to the urine resulting in fasterclearance. Accordingly, for non-kidney targets, kidney accumulation isundesired. These data suggest that GalNAc₃-1 conjugation reduces kidneyaccumulation.

TABLE 23 PK analysis of ASO treatment in transgenic mice Inter- Kid-Liver nucleoside SEQ Dose Liver ney EC₅₀ 3′ Linkage/ ID ASO (μmol/kg)(μg/g) (μg/g) (μg/g) Conjugate Length No. ISIS 0.1 5.2 2.1 53 None PS/20244 304801 0.8 62.8 119.6 2.3 142.3 191.5 6.8 202.3 337.7 ISIS 0.1 3.80.7 3.8 GalNAc₃-1 PS/20 245 647535 0.8 72.7 34.3 2.3 106.8 111.4 6.8237.2 179.3

Metabolites of ISIS 647535 were also identified and their masses wereconfirmed by high resolution mass spectrometry analysis. The cleavagesites and structures of the observed metabolites are shown below. Therelative % of full length ASO was calculated using standard proceduresand the results are presented in Table 23a. The major metabolite of ISIS647535 was full-length ASO lacking the entire conjugate (i.e. ISIS304801), which results from cleavage at cleavage site A, shown below.Further, additional metabolites resulting from other cleavage sites werealso observed. These results suggest that introducing other cleavablebonds such as esters, peptides, disulfides, phosphoramidates oracyl-hydrazones between the GalNAc₃-1 sugar and the ASO, which can becleaved by enzymes inside the cell, or which may cleave in the reductiveenvironment of the cytosol, or which are labile to the acidic pH insideendosomes and lysosomes, can also be useful.

TABLE 23a Observed full length metabolites of ISIS 647535 CleavageRelative Metabolite ASO site % 1 ISIS 304801 A 36.1 2 ISIS 304801 + dA B10.5 3 ISIS 647535 minus [3 GalNAc] C 16.1 4 ISIS 647535 minus D 17.6 [3GalNAc + 1 5-hydroxy- pentanoic acid tether] 5 ISIS 647535 minus D 9.9[2 GalNAc + 2 5-hydroxy- pentanoic acid tether] 6 ISIS 647535 minus D9.8 [3 GalNAc + 3 5-hydroxy- pentanoic acid tether]

Cleavage Sites

Example 21: Antisense Inhibition of Human ApoC III in Human ApoC IIITransgenic Mice in Single Administration Study

ISIS 304801, 647535 and 647536 each targeting human ApoC III anddescribed in Table 17, were further evaluated in a single administrationstudy for their ability to inhibit human ApoC III in human ApoC IIItransgenic mice.

Treatment

Human ApoCIII transgenic mice were maintained on a 12-hour light/darkcycle and fed ad libitum Teklad lab chow. Animals were acclimated for atleast 7 days in the research facility before initiation of theexperiment. ASOs were prepared in PBS and sterilized by filteringthrough a 0.2 micron filter. ASOs were dissolved in 0.9% PBS forinjection.

Human ApoC III transgenic mice were injected intraperitoneally once atthe dosage shown below with ISIS 304801, 647535 or 647536 (describedabove) or with PBS treated control. The treatment group consisted of 3animals and the control group consisted of 4 animals. Prior to thetreatment as well as after the last dose, blood was drawn from eachmouse and plasma samples were analyzed. The mice were sacrificed 72hours following the last administration.

Samples were collected and analyzed to determine the ApoC III mRNA andprotein levels in the liver; plasma triglycerides; and cholesterol,including HDL and LDL fractions were assessed as described above(Example 20). Data from those analyses are presented in Tables 24-28,below. Liver transaminase levels, alanine aminotransferase (ALT) andaspartate aminotransferase (AST), in serum were measured relative tosaline injected mice using standard protocols. The ALT and AST levelsshowed that the antisense compounds were well tolerated at alladministered doses.

These results show improvement in potency for antisense compoundscomprising a GalNAc₃-1 conjugate at the 3′ terminus (ISIS 647535 and647536) compared to the antisense compound lacking a GalNAc₃-1 conjugate(ISIS 304801). Further, ISIS 647536, which comprises a GalNAc₃-1conjugate and some phosphodiester linkages was as potent as ISIS 647535,which comprises the same conjugate and all internucleoside linkageswithin the ASO are phosphorothioate.

TABLE 24 Effect of ASO treatment on ApoC III mRNA levels in human ApoCIII transgenic mice Inter- nucleoside SEQ Dose % ED₅₀ 3′ linkage/ ID ASO(mg/kg) PBS (mg/kg) Conjugate Length No. PBS 0 99 — — — ISIS 1 104 13.2None PS/20 244 304801 3 92 10 71 30 40 ISIS 0.3 98 1.9 GalNAc₃-1 PS/20245 647535 1 70 3 33 10 20 ISIS 0.3 103 1.7 GalNAc₃-1 PS/PO/20 245647536 1 60 3 31 10 21

TABLE 25 Effect of ASO treatment on ApoC III plasma protein levels inhuman ApoC III transgenic mice Dose % ED₅₀ 3′ Internucleoside SEQ ID ASO(mg/kg) PBS (mg/kg) Conjugate Linkage/Length No. PBS 0 99 — — — ISIS 1104 23.2 None PS/20 244 304801 3 92 10 71 30 40 ISIS 0.3 98 2.1GalNAc₃-1 PS/20 245 647535 1 70 3 33 10 20 ISIS 0.3 103 1.8 GalNAc₃-1PS/PO/20 245 647536 1 60 3 31 10 21

TABLE 26 Effect of ASO treatment on triglyceride levels in transgenicmice Dose % ED₅₀ 3′ Internucleoside SEQ ID ASO (mg/kg) PBS (mg/kg)Conjugate Linkage/Length No. PBS 0 98 — — — ISIS 1 80 29.1 None PS/20244 304801 3 92 10 70 30 47 ISIS 0.3 100 2.2 GalNAc₃-1 PS/20 245 6475351 70 3 34 10 23 ISIS 0.3 95 1.9 GalNAc₃-1 PS/PO/20 245 647536 1 66 3 3110 23

TABLE 27 Effect of ASO treatment on total cholesterol levels intransgenic mice Dose Internucleoside ASO (mg/kg) % PBS 3′ ConjugateLinkage/Length SEQ ID No. PBS 0 96 — ISIS 1 104 None PS/20 244 304801 396 10 86 30 72 ISIS 0.3 93 GalNAc₃-1 PS/20 245 647535 1 85 3 61 10 53ISIS 0.3 115 GalNAc₃-1 PS/PO/20 245 647536 1 79 3 51 10 54

TABLE 28 Effect of ASO treatment on HDL and LDL cholesterol levels intransgenic mice HDL LDL Internucleoside SEQ Dose % % 3′ Linkage/ ID ASO(mg/kg) PBS PBS Conjugate Length No. PBS 0 131 90 — — ISIS 1 130 72 NonePS/20 244 304801 3 186 79 10 226 63 30 240 46 ISIS 0.3  98 86 GalNAc₃-1PS/20 245 647535 1 214 67 3 212 39 10 218 35 ISIS 0.3 143 89 GalNAc₃-1PS/PO/20 245 647536 1 187 56 3 213 33 10 221 34

These results confirm that the GalNAc₃-1 conjugate improves potency ofan antisense compound. The results also show equal potency of aGalNAc₃-1 conjugated antisense compounds where the antisenseoligonucleotides have mixed linkages (ISIS 647536 which has sixphosphodiester linkages) and a full phosphorothioate version of the sameantisense compound (ISIS 647535).

Phosphorothioate linkages provide several properties to antisensecompounds. For example, they resist nuclease digestion and they bindproteins resulting in accumulation of compound in the liver, rather thanin the kidney/urine. These are desirable properties, particularly whentreating an indication in the liver. However, phosphorothioate linkageshave also been associated with an inflammatory response. Accordingly,reducing the number of phosphorothioate linkages in a compound isexpected to reduce the risk of inflammation, but also lowerconcentration of the compound in liver, increase concentration in thekidney and urine, decrease stability in the presence of nucleases, andlower overall potency. The present results show that a GalNAc₃-1conjugated antisense compound where certain phosphorothioate linkageshave been replaced with phosphodiester linkages is as potent against atarget in the liver as a counterpart having full phosphorothioatelinkages. Such compounds are expected to be less proinflammatory (SeeExample 24 describing an experiment showing reduction of PS results inreduced inflammatory effect).

Example 22: Effect of GalNAc₃-1 Conjugated Modified ASO Targeting SRB-1In Vivo

ISIS 440762 and 651900, each targeting SRB-1 and described in Table 17,were evaluated in a dose-dependent study for their ability to inhibitSRB-1 in Balb/c mice.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 440762,651900 or with PBS treated control. Each treatment group consisted of 4animals. The mice were sacrificed 48 hours following the finaladministration to determine the SRB-1 mRNA levels in liver usingreal-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. SRB-1 mRNAlevels were determined relative to total RNA (using Ribogreen), prior tonormalization to PBS-treated control. The results below are presented asthe average percent of SRB-1 mRNA levels for each treatment group,normalized to PBS-treated control and is denoted as “% PBS”.

As illustrated in Table 29, both antisense compounds lowered SRB-1 mRNAlevels. Further, the antisense compound comprising the GalNAc₃-1conjugate (ISIS 651900) was substantially more potent than the antisensecompound lacking the GalNAc₃-1 conjugate (ISIS 440762). These resultsdemonstrate that the potency benefit of GalNAc₃-1 conjugates areobserved using antisense oligonucleotides complementary to a differenttarget and having different chemically modified nucleosides, in thisinstance modified nucleosides comprise constrained ethyl sugar moieties(a bicyclic sugar moiety).

TABLE 29 Effect of ASO treatment on SRB-1 mRNA levels in Balb/c mice SEQDose Liver ED₅₀ 3′ Internucleoside ID ASO (mg/kg) % PBS (mg/kg)Conjugate linkage/Length No. PBS 0 100 — — ISIS 0.7 85 2.2 None PS/14246 440762 2 55 7 12 20 3 ISIS 0.07 98 0.3 GalNAc₃-1 PS/14 247 6519000.2 63 0.7 20 2 6 7 5

Example 23: Human Peripheral Blood Mononuclear Cells (hPBMC) AssayProtocol

The hPBMC assay was performed using BD Vautainer CPT tube method. Asample of whole blood from volunteered donors with informed consent atUS HealthWorks clinic (Faraday & El Camino Real, Carlsbad) was obtainedand collected in 4-15 BD Vacutainer CPT 8 ml tubes (VWR Cat.# BD362753).The approximate starting total whole blood volume in the CPT tubes foreach donor was recorded using the PBMC assay data sheet.

The blood sample was remixed immediately prior to centrifugation bygently inverting tubes 8-10 times. CPT tubes were centrifuged at rt(18-25° C.) in a horizontal (swing-out) rotor for 30 min. at 1500-1800RCF with brake off (2700 RPM Beckman Allegra 6R). The cells wereretrieved from the buffy coat interface (between Ficoll and polymer gellayers); transferred to a sterile 50 ml conical tube and pooled up to 5CPT tubes/50 ml conical tube/donor. The cells were then washed twicewith PBS (Ca⁺⁺, Mg⁺⁺ free; GIBCO). The tubes were topped up to 50 ml andmixed by inverting several times. The sample was then centrifuged at330×g for 15 minutes at rt (1215 RPM in Beckman Allegra 6R) andaspirated as much supernatant as possible without disturbing pellet. Thecell pellet was dislodged by gently swirling tube and resuspended cellsin RPMI+10% FBS+pen/strep (˜1 ml/10 ml starting whole blood volume). A60 μl sample was pipette into a sample vial (Beckman Coulter) with 600μl VersaLyse reagent (Beckman Coulter Cat# A09777) and was gentlyvortexed for 10-15 sec. The sample was allowed to incubate for 10 min.at rt and being mixed again before counting. The cell suspension wascounted on Vicell XR cell viability analyzer (Beckman Coulter) usingPBMC cell type (dilution factor of 1:11 was stored with otherparameters). The live cell/ml and viability were recorded. The cellsuspension was diluted to 1×10⁷ live PBMC/ml in RPMI+10% FBS+pen/strep.

The cells were plated at 5×10⁵ in 50 μl/well of 96-well tissue cultureplate (Falcon Microtest). 50 μl/well of 2× concentration oligos/controlsdiluted in RPMI+10% FBS+pen/strep. was added according to experimenttemplate (100 μl/well total). Plates were placed on the shaker andallowed to mix for approx. 1 min. After being incubated for 24 hrs at37° C.; 5% CO₂, the plates were centrifuged at 400×g for 10 minutesbefore removing the supernatant for MSD cytokine assay (i.e. human IL-6,IL-10, IL-8 and MCP-1).

Example 24: Evaluation of Proinflammatory Effects in hPBMC Assay forGalNAc₃-1 Conjugated ASOs

The antisense oligonucleotides (ASOs) listed in Table 30 were evaluatedfor proinflammatory effect in hPBMC assay using the protocol describedin Example 23. ISIS 353512 is an internal standard known to be a highresponder for IL-6 release in the assay. The hPBMCs were isolated fromfresh, volunteered donors and were treated with ASOs at 0, 0.0128,0.064, 0.32, 1.6, 8, 40 and 200 μM concentrations. After a 24 hrtreatment, the cytokine levels were measured.

The levels of IL-6 were used as the primary readout. The EC₅₀ andE_(max) was calculated using standard procedures. Results are expressedas the average ratio of E_(max)/EC₅₀ from two donors and is denoted as“E_(max)/EC₅₀.” The lower ratio indicates a relative decrease in theproinflammatory response and the higher ratio indicates a relativeincrease in the proinflammatory response.

With regard to the test compounds, the least proinflammatory compoundwas the PS/PO linked ASO (ISIS 616468). The GalNAc₃-1 conjugated ASO,ISIS 647535 was slightly less proinflammatory than its non-conjugatedcounterpart ISIS 304801. These results indicate that incorporation ofsome PO linkages reduces proinflammatory reaction and addition of aGalNAc₃-1 conjugate does not make a compound more proinflammatory andmay reduce proinflammatory response. Accordingly, one would expect thatan antisense compound comprising both mixed PS/PO linkages and aGalNAc₃-1 conjugate would produce lower proinflammatory responsesrelative to full PS linked antisense compound with or without aGalNAc₃-1 conjugate. These results show that GalNAc₃-1 conjugatedantisense compounds, particularly those having reduced PS content areless proinflammatory.

Together, these results suggest that a GalNAc₃-1 conjugated compound,particularly one with reduced PS content, can be administered at ahigher dose than a counterpart full PS antisense compound lacking aGalNAc₃-1 conjugate. Since half-life is not expected to be substantiallydifferent for these compounds, such higher administration would resultin less frequent dosing. Indeed such administration could be even lessfrequent, because the GalNAc₃-1 conjugated compounds are more potent(See Examples 20-22) and re-dosing is necessary once the concentrationof a compound has dropped below a desired level, where such desiredlevel is based on potency.

TABLE 30 Modified ASOs SEQ ID ASO Sequence (5′ to 3′) Target No. ISISG_(es) ^(m)C_(es)T_(es)G_(es)A_(es)T_(ds)T_(ds)A_(ds)G_(ds)A_(ds)G_(ds)TNFα 248 104838 A_(ds)G_(ds)A_(ds)G_(ds)G_(es)T_(es) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) ISIS T_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(ds)A_(ds)T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds) CRP 249 353512G_(ds)A_(ds)G_(ds)A_(ds) ^(m)C_(ds) ^(m)C_(ds)T_(es)G_(es)G_(e) ISISA_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds)ApoC III 244 304801 ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) ISIS A_(es)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ApoC III 245647535 ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) ISISA_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds)ApoC III 244 616468 ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(e)

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicatesβ-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside(e.g. cEt); “s” indicates phosphorothioate internucleoside linkages(PS); “o” indicates phosphodiester internucleoside linkages (PO); and“o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates5-methylcytosines. “A_(do′)-GalNAc₃-1_(a)” indicates a conjugate havingthe structure GalNAc₃-1 shown in Example 9 attached to the 3′-end of theantisense oligonucleotide, as indicated.

TABLE 31 Proinflammatory Effect of ASOs targeting ApoC III in hPBMCassay Internucleoside SEQ EC₅₀ E_(max) E_(max)/ 3′ Linkage/ ID ASO (μM)(μM) EC₅₀ Conjugate Length No. ISIS 353512 0.01 265.9 26,590 None PS/20249 (high responder) ISIS 304801 0.07 106.55 1,522 None PS/20 244 ISIS647535 0.12 138 1,150 GalNAc₃-1 PS/20 245 ISIS 616468 0.32 71.52 224None PS/PO/20 244

Example 25: Effect of GalNAc₃-1 Conjugated Modified ASO Targeting HumanApoC III In Vitro

ISIS 304801 and 647535 described above were tested in vitro. Primaryhepatocyte cells from transgenic mice at a density of 25,000 cells perwell were treated with 0.03, 0.08, 0.24, 0.74, 2.22, 6.67 and 20 μMconcentrations of modified oligonucleotides. After a treatment period ofapproximately 16 hours, RNA was isolated from the cells and mRNA levelswere measured by quantitative real-time PCR and the hApoC III mRNAlevels were adjusted according to total RNA content, as measured byRIBOGREEN.

The IC₅₀ was calculated using the standard methods and the results arepresented in Table 32. As illustrated, comparable potency was observedin cells treated with ISIS 647535 as compared to the control, ISIS304801.

TABLE 32 Modified ASO targeting human ApoC III in primary hepatocytesInternucleoside SEQ ASO IC₅₀ (μM) 3′ Conjugate linkage/Length ID No.ISIS 0.44 None PS/20 244 304801 ISIS 0.31 GalNAc₃-1 PS/20 245 647535

In this experiment, the large potency benefits of GalNAc₃-1 conjugationthat are observed in vivo were not observed in vitro. Subsequent freeuptake experiments in primary hepatocytes in vitro did show increasedpotency of oligonucleotides comprising various GalNAc conjugatesrelative to oligonucleotides that lacking the GalNAc conjugate. (seeExamples 60, 82, and 92)

Example 26: Effect of PO/PS Linkages on ApoC III ASO Activity

Human ApoC III transgenic mice were injected intraperitoneally once at25 mg/kg of ISIS 304801, or ISIS 616468 (both described above) or withPBS treated control once per week for two weeks. The treatment groupconsisted of 3 animals and the control group consisted of 4 animals.Prior to the treatment as well as after the last dose, blood was drawnfrom each mouse and plasma samples were analyzed. The mice weresacrificed 72 hours following the last administration.

Samples were collected and analyzed to determine the ApoC III proteinlevels in the liver as described above (Example 20). Data from thoseanalyses are presented in Table 33, below.

These results show reduction in potency for antisense compounds withPO/PS (ISIS 616468) in the wings relative to full PS (ISIS 304801).

TABLE 33 Effect of ASO treatment on ApoC III protein levels in humanApoC III transgenic mice Dose 3′ Internucleoside SEQ ID ASO (mg/kg) %PBS Conjugate linkage/Length No. PBS  0 99 — — ISIS 25 24 None Full PS244 304801 mg/kg/wk for 2 wks ISIS 25 40 None 14 PS/6 PO 244 616468mg/kg/wk for 2 wks

Example 27: Compound 56

Compound 56 is commercially available from Glen Research or may beprepared according to published procedures reported by Shchepinov etal., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 28: Preparation of Compound 60

Compound 4 was prepared as per the procedures illustrated in Example 2.Compound 57 is commercially available. Compound 60 was confirmed bystructural analysis.

Compound 57 is meant to be representative and not intended to belimiting as other monoprotected substituted or unsubstituted alkyl diolsincluding but not limited to those presented in the specification hereincan be used to prepare phosphoramidites having a predeterminedcomposition.

Example 29: Preparation of Compound 63

Compounds 61 and 62 are prepared using procedures similar to thosereported by Tober et al., Eur. J. Org. Chem., 2013, 3, 566-577; andJiang et al., Tetrahedron, 2007, 63(19), 3982-3988.

Alternatively, Compound 63 is prepared using procedures similar to thosereported in scientific and patent literature by Kim et al., Synlett,2003, 12, 1838-1840; and Kim et al., published PCT InternationalApplication, WO 2004063208.

Example 30: Preparation of Compound 63b

Compound 63a is prepared using procedures similar to those reported byHanessian et al., Canadian Journal of Chemistry, 1996, 74(9), 1731-1737.

Example 31: Preparation of Compound 63d

Compound 63c is prepared using procedures similar to those reported byChen et al., Chinese Chemical Letters, 1998, 9(5), 451-453.

Example 32: Preparation of Compound 67

Compound 64 was prepared as per the procedures illustrated in Example 2.Compound 65 is prepared using procedures similar to those reported by Oret al., published PCT International Application, WO 2009003009. Theprotecting groups used for Compound 65 are meant to be representativeand not intended to be limiting as other protecting groups including butnot limited to those presented in the specification herein can be used.

Example 33: Preparation of Compound 70

Compound 64 was prepared as per the procedures illustrated in Example 2.Compound 68 is commercially available. The protecting group used forCompound 68 is meant to be representative and not intended to belimiting as other protecting groups including but not limited to thosepresented in the specification herein can be used.

Example 34: Preparation of Compound 75a

Compound 75 is prepared according to published procedures reported byShchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 35: Preparation of Compound 79

Compound 76 was prepared according to published procedures reported byShchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 36: Preparation of Compound 79a

Compound 77 is prepared as per the procedures illustrated in Example 35.

Example 37: General Method for the Preparation of Conjugated OligomericCompound 82 Comprising a Phosphodiester Linked GalNAc₃-2 Conjugate at 5′Terminus Via Solid Support (Method I)

wherein GalNAc₃-2 has the structure:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-2(GalNAc₃-2_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. Wherein GalNAc₃-2_(a) has the formula:

The VIMAD-bound oligomeric compound 79b was prepared using standardprocedures for automated DNA/RNA synthesis (see Dupouy et al., Angew.Chem. Int. Ed., 2006, 45, 3623-3627). The phosphoramidite Compounds 56and 60 were prepared as per the procedures illustrated in Examples 27and 28, respectively. The phosphoramidites illustrated are meant to berepresentative and not intended to be limiting as other phosphoramiditebuilding blocks including but not limited those presented in thespecification herein can be used to prepare an oligomeric compoundhaving a phosphodiester linked conjugate group at the 5′ terminus. Theorder and quantity of phosphoramidites added to the solid support can beadjusted to prepare the oligomeric compounds as described herein havingany predetermined sequence and composition.

Example 38: Alternative Method for the Preparation of OligomericCompound 82 Comprising a Phosphodiester Linked GalNAc₃-2 Conjugate at 5′Terminus (Method II)

The VIMAD-bound oligomeric compound 79b was prepared using standardprocedures for automated DNA/RNA synthesis (see Dupouy et al., Angew.Chem. Int. Ed., 2006, 45, 3623-3627). The GalNAc₃-2 clusterphosphoramidite, Compound 79 was prepared as per the proceduresillustrated in Example 35. This alternative method allows a one-stepinstallation of the phosphodiester linked GalNAc₃-2 conjugate to theoligomeric compound at the final step of the synthesis. Thephosphoramidites illustrated are meant to be representative and notintended to be limiting, as other phosphoramidite building blocksincluding but not limited to those presented in the specification hereincan be used to prepare oligomeric compounds having a phosphodiesterconjugate at the 5′ terminus. The order and quantity of phosphoramiditesadded to the solid support can be adjusted to prepare the oligomericcompounds as described herein having any predetermined sequence andcomposition.

Example 39: General Method for the Preparation of Oligomeric Compound83h Comprising a GalNAc₃-3 Conjugate at the 5′ Terminus (GalNAc₃-1Modified for 5′ End Attachment) Via Solid Support

Compound 18 was prepared as per the procedures illustrated in Example 4.Compounds 83a and 83b are commercially available. Oligomeric Compound83e comprising a phosphodiester linked hexylamine was prepared usingstandard oligonucleotide synthesis procedures. Treatment of theprotected oligomeric compound with aqueous ammonia provided the5′-GalNAc₃-3 conjugated oligomeric compound (83h).

Wherein GalNAc₃-3 has the structure:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-3(GalNAc₃-3_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. Wherein GalNAc₃-3_(a) has the formula:

Example 40: General Method for the Preparation of Oligomeric Compound 89Comprising a Phosphodiester Linked GalNAc₃-4 Conjugate at the 3′Terminus Via Solid Support

Wherein GalNAc₃-4 has the structure:

Wherein CM is a cleavable moiety. In certain embodiments, cleavablemoiety is:

The GalNAc₃ cluster portion of the conjugate group GalNAc₃-4(GalNAc₃-4_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. Wherein GalNAc₃-4_(a) has the formula:

The protected Unylinker functionalized solid support Compound 30 iscommercially available. Compound 84 is prepared using procedures similarto those reported in the literature (see Shchepinov et al., NucleicAcids Research, 1997, 25(22), 4447-4454; Shchepinov et al., NucleicAcids Research, 1999, 27, 3035-3041; and Hornet et al., Nucleic AcidsResearch, 1997, 25, 4842-4849).

The phosphoramidite building blocks, Compounds 60 and 79a are preparedas per the procedures illustrated in Examples 28 and 36. Thephosphoramidites illustrated are meant to be representative and notintended to be limiting as other phosphoramidite building blocks can beused to prepare an oligomeric compound having a phosphodiester linkedconjugate at the 3′ terminus with a predetermined sequence andcomposition. The order and quantity of phosphoramidites added to thesolid support can be adjusted to prepare the oligomeric compounds asdescribed herein having any predetermined sequence and composition.

Example 41: General Method for the Preparation of ASOs Comprising aPhosphodiester Linked GalNAc₃-2 (See Example 37, Bx is Adenine)Conjugate at the 5′ Position Via Solid Phase Techniques (Preparation ofISIS 661134)

Unless otherwise stated, all reagents and solutions used for thesynthesis of oligomeric compounds are purchased from commercial sources.Standard phosphoramidite building blocks and solid support are used forincorporation nucleoside residues which include for example T, A, G, and^(m)C residues. Phosphoramidite compounds 56 and 60 were used tosynthesize the phosphodiester linked GalNAc₃-2 conjugate at the 5′terminus. A 0.1 M solution of phosphoramidite in anhydrous acetonitrilewas used for β-D-2′-deoxyribonucleoside and 2′-MOE.

The ASO syntheses were performed on ABI 394 synthesizer (1-2 μmol scale)or on GE Healthcare Bioscience ÄKTA oligopilot synthesizer (40-200 μmolscale) by the phosphoramidite coupling method on VIMAD solid support(110 μmol/g, Guzaev et al., 2003) packed in the column. For the couplingstep, the phosphoramidites were delivered at a 4 fold excess over theinitial loading of the solid support and phosphoramidite coupling wascarried out for 10 min. All other steps followed standard protocolssupplied by the manufacturer. A solution of 6% dichloroacetic acid intoluene was used for removing the dimethoxytrityl (DMT) groups from5′-hydroxyl groups of the nucleotide. 4,5-Dicyanoimidazole (0.7 M) inanhydrous CH₃CN was used as activator during the coupling step.Phosphorothioate linkages were introduced by sulfurization with 0.1 Msolution of xanthane hydride in 1:1 pyridine/CH₃CN for a contact time of3 minutes. A solution of 20% tert-butylhydroperoxide in CH₃CN containing6% water was used as an oxidizing agent to provide phosphodiesterinternucleoside linkages with a contact time of 12 minutes.

After the desired sequence was assembled, the cyanoethyl phosphateprotecting groups were deprotected using a 20% diethylamine in toluene(v/v) with a contact time of 45 minutes. The solid-support bound ASOswere suspended in aqueous ammonia (28-30 wt %) and heated at 55° C. for6 h. The unbound ASOs were then filtered and the ammonia was boiled off.The residue was purified by high pressure liquid chromatography on astrong anion exchange column (GE Healthcare Bioscience, Source 30Q, 30μm, 2.54×8 cm, A=100 mM ammonium acetate in 30% aqueous CH₃CN, B=1.5 MNaBr in A, O-40% of B in 60 min, flow 14 mL min-1, λ=260 nm). Theresidue was desalted by HPLC on a reverse phase column to yield thedesired ASOs in an isolated yield of 15-30% based on the initial loadingon the solid support. The ASOs were characterized by ion-pair-HPLCcoupled MS analysis with Agilent 1100 MSD system.

TABLE 34 ASO comprising a phosphodiester linked GalNAc₃-2 conjugate atthe 5′ position targeting SRB-1 Observed SEQ ID ISIS No. Sequence (5′ to3′) CalCd Mass Mass No. 661134 GalNAc ₃ -2 _(a) - _(o′) A _(do)T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) 6482.2 6481.6 250G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k)

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicatesβ-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside(e.g. cEt); “s” indicates phosphorothioate internucleoside linkages(PS); “o” indicates phosphodiester internucleoside linkages (PO); and“o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates5-methylcytosines. The structure of GalNAc₃-2_(a) is shown in Example37.

Example 42: General Method for the Preparation of ASOs Comprising aGalNAc₃-3 Conjugate at the 5′ Position Via Solid Phase Techniques(Preparation of ISIS 661166)

The synthesis for ISIS 661166 was performed using similar procedures asillustrated in Examples 39 and 41.

ISIS 661166 is a 5-10-5 MOE gapmer, wherein the 5′ position comprises aGalNAc₃-3 conjugate. The ASO was characterized by ion-pair-HPLC coupledMS analysis with Agilent 1100 MSD system.

TABLE 34a ASO comprising a GalNAc₃-3 conjugate at the 5′ position via ahexylamino phosphodiester linkage targeting Malat-1 ISIS Calcd ObservedNo. Sequence (5′ to 3′) Conjugate Mass Mass SEQ ID No. 661166 5′-GalNAc₃ -3 _(a-o′) ^(m)C_(es)G_(es)G_(es)T_(es)G_(es) 5′-GalNAc ₃ -3 8992.168990.51 251 ^(m)C_(ds)A_(ds)A_(ds)G_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(ds)A_(ds)G_(ds) G_(es)A_(es)A_(es)T_(es)T_(e)

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicatesβ-D-2′-deoxyribonucleoside; “s” indicates phosphorothioateinternucleoside linkages (PS); “o” indicates phosphodiesterinternucleoside linkages (PO); and “o′” indicates —O—P(═O)(OH)—.Superscript “m” indicates 5-methylcytosines. The structure of“5′-GalNAc₃-3a” is shown in Example 39.

Example 43: Dose-Dependent Study of Phosphodiester Linked GalNAc₃-2 (SeeExamples 37 and 41, Bx is Adenine) at the 5′ Terminus Targeting SRB-1 InVivo

ISIS 661134 (see Example 41) comprising a phosphodiester linkedGalNAc₃-2 conjugate at the 5′ terminus was tested in a dose-dependentstudy for antisense inhibition of SRB-1 in mice. Unconjugated ISIS440762 and 651900 (GalNAc₃-1 conjugate at 3′ terminus, see Example 9)were included in the study for comparison and are described previouslyin Table 17.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 440762,651900, 661134 or with PBS treated control. Each treatment groupconsisted of 4 animals. The mice were sacrificed 72 hours following thefinal administration to determine the liver SRB-1 mRNA levels usingreal-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. SRB-1 mRNAlevels were determined relative to total RNA (using Ribogreen), prior tonormalization to PBS-treated control. The results below are presented asthe average percent of SRB-1 mRNA levels for each treatment group,normalized to PBS-treated control and is denoted as “% PBS”. The ED₅₀swere measured using similar methods as described previously and arepresented below.

As illustrated in Table 35, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, theantisense oligonucleotides comprising the phosphodiester linkedGalNAc₃-2 conjugate at the 5′ terminus (ISIS 661134) or the GalNAc₃-1conjugate linked at the 3′ terminus (ISIS 651900) showed substantialimprovement in potency compared to the unconjugated antisenseoligonucleotide (ISIS 440762). Further, ISIS 661134, which comprises thephosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus wasequipotent compared to ISIS 651900, which comprises the GalNAc₃-1conjugate at the 3′ terminus.

TABLE 35 ASOs containing GalNAc₃-1 or GalNAc₃-2 targeting SRB-1 ISISDosage SRB-1 mRNA ED₅₀ No. (mg/kg) levels (% PBS) (mg/kg) Conjugate SEQID No. PBS 0 100 — — 440762 0.2 116 2.58 No conjugate 250 0.7 91 2 69 722 20 5 651900 0.07 95 0.26 3′ GalNAc₃-1 247 0.2 77 0.7 28 2 11 7 8661134 0.07 107 0.25 5′ GalNAc₃-2 250 0.2 86 0.7 28 2 10 7 6 Structuresfor 3′ GalNAc₃-1 and 5′ GalNAc₃-2 were described previously in Examples9 and 37.

Pharmacokinetics Analysis (PK)

The PK of the ASOs from the high dose group (7 mg/kg) was examined andevaluated in the same manner as illustrated in Example 20. Liver samplewas minced and extracted using standard protocols. The full lengthmetabolites of 661134 (5′ GalNAc₃-2) and ISIS 651900 (3′ GalNAc₃-1) wereidentified and their masses were confirmed by high resolution massspectrometry analysis. The results showed that the major metabolitedetected for the ASO comprising a phosphodiester linked GalNAc₃-2conjugate at the 5′ terminus (ISIS 661134) was ISIS 440762 (data notshown). No additional metabolites, at a detectable level, were observed.Unlike its counterpart, additional metabolites similar to those reportedpreviously in Table 23a were observed for the ASO having the GalNAc₃-1conjugate at the 3′ terminus (ISIS 651900). These results suggest thathaving the phosphodiester linked GalNAc₃-1 or GalNAc₃-2 conjugate mayimprove the PK profile of ASOs without compromising their potency.

Example 44: Effect of PO/PS Linkages on Antisense Inhibition of ASOsComprising GalNAc₃-1 Conjugate (See Example 9) at the 3′ TerminusTargeting SRB-1

ISIS 655861 and 655862 comprising a GalNAc₃-1 conjugate at the 3′terminus each targeting SRB-1 were tested in a single administrationstudy for their ability to inhibit SRB-1 in mice. The parentunconjugated compound, ISIS 353382 was included in the study forcomparison.

The ASOs are 5-10-5 MOE gapmers, wherein the gap region comprises ten2′-deoxyribonucleosides and each wing region comprises five 2′-MOEmodified nucleosides. The ASOs were prepared using similar methods asillustrated previously in Example 19 and are described Table 36, below.

TABLE 36 Modified ASOs comprising GalNAc₃-1 conjugate at the 3′ terminustargeting SRB-1 SEQ ID ISIS No. Sequence (5′ to 3′) Chemistry No. 353382G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) Full PS no conjugate 252 (parent)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 655861 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) Full PS with 253^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃ -1 _(a) GalNAc ₃ -1 conjugate 655862 G_(es)^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) Mixed PS/PO with 253^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃ -1 _(a) GalNAc ₃ -1 conjugate

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicatesβ-D-2′-deoxyribonucleoside; “s” indicates phosphorothioateinternucleoside linkages (PS); “o” indicates phosphodiesterinternucleoside linkages (PO); and “o′” indicates —O—P(═O)(OH)—.Superscript “m” indicates 5-methylcytosines. The structure of“GalNAc₃-1” is shown in Example 9.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 353382,655861, 655862 or with PBS treated control. Each treatment groupconsisted of 4 animals. Prior to the treatment as well as after the lastdose, blood was drawn from each mouse and plasma samples were analyzed.The mice were sacrificed 72 hours following the final administration todetermine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN®RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.)according to standard protocols. SRB-1 mRNA levels were determinedrelative to total RNA (using Ribogreen), prior to normalization toPBS-treated control. The results below are presented as the averagepercent of SRB-1 mRNA levels for each treatment group, normalized toPBS-treated control and is denoted as “% PBS”. The ED₅₀s were measuredusing similar methods as described previously and are reported below.

As illustrated in Table 37, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner compared to PBStreated control. Indeed, the antisense oligonucleotides comprising theGalNAc₃-1 conjugate at the 3′ terminus (ISIS 655861 and 655862) showedsubstantial improvement in potency comparing to the unconjugatedantisense oligonucleotide (ISIS 353382). Further, ISIS 655862 with mixedPS/PO linkages showed an improvement in potency relative to full PS(ISIS 655861).

TABLE 37 Effect of PO/PS linkages on antisense inhibition of ASOscomprising GalNAc₃-1 conjugate at 3′ terminus targeting SRB-1 ISISDosage SRB-1 mRNA ED₅₀ SEQ No. (mg/kg) levels (% PBS) (mg/kg) ChemistryID No. PBS 0 100 — — 353382 3 76.65 10.4 Full PS 252 (parent) 10 52.40without 30 24.95 conjugate 655861 0.5 81.22 2.2 Full PS with 253 1.563.51 GalNAc₃-1 5 24.61 conjugate 15 14.80 655862 0.5 69.57 1.3 MixedPS/PO 253 1.5 45.78 with 5 19.70 GalNAc₃-1 15 12.90 conjugate

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Organ weights were alsoevaluated. The results demonstrated that no elevation in transaminaselevels (Table 38) or organ weights (data not shown) were observed inmice treated with ASOs compared to PBS control. Further, the ASO withmixed PS/PO linkages (ISIS 655862) showed similar transaminase levelscompared to full PS (ISIS 655861).

TABLE 38 Effect of PO/PS linkages on transaminase levels of ASOscomprising GalNAc₃-1 conjugate at 3′ terminus targeting SRB-1 ISISDosage ALT AST No. (mg/kg) (U/L) (U/L) Chemistry SEQ ID No. PBS 0 28.565 — 353382 3 50.25 89 Full PS without 252 (parent) 10 27.5 79.3conjugate 30 27.3 97 655861 0.5 28 55.7 Full PS with 253 1.5 30 78GalNAc₃-1 5 29 63.5 15 28.8 67.8 655862 0.5 50 75.5 Mixed PS/PO with 2531.5 21.7 58.5 GalNAc₃-1 5 29.3 69 15 22 61

Example 45: Preparation of PFP Ester, Compound 110a

Compound 4 (9.5 g, 28.8 mmoles) was treated with compound 103a or 103b(38 mmoles), individually, and TMSOTf (0.5 eq.) and molecular sieves indichloromethane (200 mL), and stirred for 16 hours at room temperature.At that time, the organic layer was filtered thru celite, then washedwith sodium bicarbonate, water and brine. The organic layer was thenseparated and dried over sodium sulfate, filtered and reduced underreduced pressure. The resultant oil was purified by silica gelchromatography (2%-->10% methanol/dichloromethane) to give compounds104a and 104b in >80% yield. LCMS and proton NMR was consistent with thestructure.

Compounds 104a and 104b were treated to the same conditions as forcompounds 100a-d (Example 47), to give compounds 105a and 105b in >90%yield. LCMS and proton NMR was consistent with the structure.

Compounds 105a and 105b were treated, individually, with compound 90under the same conditions as for compounds 901a-d, to give compounds106a (80%) and 106b (20%). LCMS and proton NMR was consistent with thestructure.

Compounds 106a and 106b were treated to the same conditions as forcompounds 96a-d (Example 47), to give 107a (60%) and 107b (20%). LCMSand proton NMR was consistent with the structure.

Compounds 107a and 107b were treated to the same conditions as forcompounds 97a-d (Example 47), to give compounds 108a and 108b in 40-60%yield. LCMS and proton NMR was consistent with the structure.

Compounds 108a (60%) and 108b (40%) were treated to the same conditionsas for compounds 100a-d (Example 47), to give compounds 109a and 109bin >80% yields. LCMS and proton NMR was consistent with the structure.

Compound 109a was treated to the same conditions as for compounds 101a-d(Example 47), to give Compound 110a in 30-60% yield. LCMS and proton NMRwas consistent with the structure. Alternatively, Compound 110b can beprepared in a similar manner starting with Compound 109b.

Example 46: General Procedure for Conjugation with PFP Esters(Oligonucleotide 111); Preparation of ISIS 666881 (GalNAc₃-10)

A 5′-hexylamino modified oligonucleotide was synthesized and purifiedusing standard solid-phase oligonucleotide procedures. The 5′-hexylaminomodified oligonucleotide was dissolved in 0.1 M sodium tetraborate, pH8.5 (200 μL) and 3 equivalents of a selected PFP esterified GalNAc₃cluster dissolved in DMSO (50 μL) was added. If the PFP esterprecipitated upon addition to the ASO solution DMSO was added until allPFP ester was in solution. The reaction was complete after about 16 h ofmixing at room temperature. The resulting solution was diluted withwater to 12 mL and then spun down at 3000 rpm in a spin filter with amass cut off of 3000 Da. This process was repeated twice to remove smallmolecule impurities. The solution was then lyophilized to dryness andredissolved in concentrated aqueous ammonia and mixed at roomtemperature for 2.5 h followed by concentration in vacuo to remove mostof the ammonia. The conjugated oligonucleotide was purified and desaltedby RP-HPLC and lyophilized to provide the GalNAc₃ conjugatedoligonucleotide.

Oligonucleotide 111 is conjugated with GalNAc₃-10. The GalNAc₃ clusterportion of the conjugate group GalNAc₃-10 (GalNAc₃-10_(a)) can becombined with any cleavable moiety to provide a variety of conjugategroups. In certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)— as shown in the oligonucleotide (ISIS666881) synthesized with GalNAc₃-10 below. The structure of GalNAc₃-10(GalNAc₃-10_(a)-CM-) is shown below:

Following this general procedure ISIS 666881 was prepared. 5′-hexylaminomodified oligonucleotide, ISIS 660254, was synthesized and purifiedusing standard solid-phase oligonucleotide procedures. ISIS 660254 (40mg, 5.2 μmop was dissolved in 0.1 M sodium tetraborate, pH 8.5 (200 μL)and 3 equivalents PFP ester (Compound 110a) dissolved in DMSO (50 μL)was added. The PFP ester precipitated upon addition to the ASO solutionrequiring additional DMSO (600 μL) to fully dissolve the PFP ester. Thereaction was complete after 16 h of mixing at room temperature. Thesolution was diluted with water to 12 mL total volume and spun down at3000 rpm in a spin filter with a mass cut off of 3000 Da. This processwas repeated twice to remove small molecule impurities. The solution waslyophilized to dryness and redissolved in concentrated aqueous ammoniawith mixing at room temperature for 2.5 h followed by concentration invacuo to remove most of the ammonia. The conjugated oligonucleotide waspurified and desalted by RP-HPLC and lyophilized to give ISIS 666881 in90% yield by weight (42 mg, 4.7 μmol).

GalNAc₃-10 conjugated oligonucleotide SEQ ASO Sequence (5′ to 3′)5′ group ID No. ISIS 660254 NH₂(CH₂)₆-_(o)A_(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) Hexylamine 254^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(es)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666881 GalNAc ₃ -10 _(a) - _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) GalNAc ₃-10 254 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

Example 47: Preparation of Oligonucleotide 102 Comprising GalNAc₃-8

The triacid 90 (4 g, 14.43 mmol) was dissolved in DMF (120 mL) andN,N-Diisopropylethylamine (12.35 mL, 72 mmoles). Pentafluorophenyltrifluoroacetate (8.9 mL, 52 mmoles) was added dropwise, under argon,and the reaction was allowed to stir at room temperature for 30 minutes.Boc-diamine 91a or 91b (68.87 mmol) was added, along withN,N-Diisopropylethylamine (12.35 mL, 72 mmoles), and the reaction wasallowed to stir at room temperature for 16 hours. At that time, the DMFwas reduced by >75% under reduced pressure, and then the mixture wasdissolved in dichloromethane. The organic layer was washed with sodiumbicarbonate, water and brine. The organic layer was then separated anddried over sodium sulfate, filtered and reduced to an oil under reducedpressure. The resultant oil was purified by silica gel chromatography(2%-->10% methanol/dichloromethane) to give compounds 92a and 92b in anapproximate 80% yield. LCMS and proton NMR were consistent with thestructure.

Compound 92a or 92b (6.7 mmoles) was treated with 20 mL ofdichloromethane and 20 mL of trifluoroacetic acid at room temperaturefor 16 hours. The resultant solution was evaporated and then dissolvedin methanol and treated with DOWEX-OH resin for 30 minutes. Theresultant solution was filtered and reduced to an oil under reducedpressure to give 85-90% yield of compounds 93a and 93b.

Compounds 7 or 64 (9.6 mmoles) were treated with HBTU (3.7 g, 9.6mmoles) and N,N-Diisopropylethylamine (5 mL) in DMF (20 mL) for 15minutes. To this was added either compounds 93a or 93b (3 mmoles), andallowed to stir at room temperature for 16 hours. At that time, the DMFwas reduced by >75% under reduced pressure, and then the mixture wasdissolved in dichloromethane. The organic layer was washed with sodiumbicarbonate, water and brine. The organic layer was then separated anddried over sodium sulfate, filtered and reduced to an oil under reducedpressure. The resultant oil was purified by silica gel chromatography(5%-->20% methanol/dichloromethane) to give compounds 96a-d in 20-40%yield. LCMS and proton NMR was consistent with the structure.

Compounds 96a-d (0.75 mmoles), individually, were hydrogenated overRaney Nickel for 3 hours in Ethanol (75 mL). At that time, the catalystwas removed by filtration thru celite, and the ethanol removed underreduced pressure to give compounds 97a-d in 80-90% yield. LCMS andproton NMR were consistent with the structure.

Compound 23 (0.32 g, 0.53 mmoles) was treated with HBTU (0.2 g, 0.53mmoles) and N,N-Diisopropylethylamine (0.19 mL, 1.14 mmoles) in DMF (30mL) for 15 minutes. To this was added compounds 97a-d (0.38 mmoles),individually, and allowed to stir at room temperature for 16 hours. Atthat time, the DMF was reduced by >75% under reduced pressure, and thenthe mixture was dissolved in dichloromethane. The organic layer waswashed with sodium bicarbonate, water and brine. The organic layer wasthen separated and dried over sodium sulfate, filtered and reduced to anoil under reduced pressure. The resultant oil was purified by silica gelchromatography (2%-->20% methanol/dichloromethane) to give compounds98a-d in 30-40% yield. LCMS and proton NMR was consistent with thestructure.

Compound 99 (0.17 g, 0.76 mmoles) was treated with HBTU (0.29 g, 0.76mmoles) and N,N-Diisopropylethylamine (0.35 mL, 2.0 mmoles) in DMF (50mL) for 15 minutes. To this was added compounds 97a-d (0.51 mmoles),individually, and allowed to stir at room temperature for 16 hours. Atthat time, the DMF was reduced by >75% under reduced pressure, and thenthe mixture was dissolved in dichloromethane. The organic layer waswashed with sodium bicarbonate, water and brine. The organic layer wasthen separated and dried over sodium sulfate, filtered and reduced to anoil under reduced pressure. The resultant oil was purified by silica gelchromatography (5%-->20% methanol/dichloromethane) to give compounds100a-d in 40-60% yield. LCMS and proton NMR was consistent with thestructure.

Compounds 100a-d (0.16 mmoles), individually, were hydrogenated over 10%Pd(OH)₂/C for 3 hours in methanol/ethyl acetate (1:1, 50 mL). At thattime, the catalyst was removed by filtration thru celite, and theorganics removed under reduced pressure to give compounds 101a-d in80-90% yield. LCMS and proton NMR was consistent with the structure.

Compounds 101a-d (0.15 mmoles), individually, were dissolved in DMF (15mL) and pyridine (0.016 mL, 0.2 mmoles). Pentafluorophenyltrifluoroacetate (0.034 mL, 0.2 mmoles) was added dropwise, under argon,and the reaction was allowed to stir at room temperature for 30 minutes.At that time, the DMF was reduced by >75% under reduced pressure, andthen the mixture was dissolved in dichloromethane. The organic layer waswashed with sodium bicarbonate, water and brine. The organic layer wasthen separated and dried over sodium sulfate, filtered and reduced to anoil under reduced pressure. The resultant oil was purified by silica gelchromatography (2%-->5% methanol/dichloromethane) to give compounds102a-d in an approximate 80% yield. LCMS and proton NMR were consistentwith the structure.

Oligomeric Compound 102, comprising a GalNAc₃-8 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-8 (GalNAc₃-8_(a))can be combined with any cleavable moiety to provide a variety ofconjugate groups. In a preferred embodiment, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-8 (GalNAc₃-8_(a)-CM-) is shown below:

Example 48: Preparation of Oligonucleotide 119 Comprising GalNAc₃-7

Compound 112 was synthesized following the procedure described in theliterature Med. Chem. 2004, 47, 5798-5808).

Compound 112 (5 g, 8.6 mmol) was dissolved in 1:1 methanol/ethyl acetate(22 mL/22 mL). Palladium hydroxide on carbon (0.5 g) was added. Thereaction mixture was stirred at room temperature under hydrogen for 12h. The reaction mixture was filtered through a pad of celite and washedthe pad with 1:1 methanol/ethyl acetate. The filtrate and the washingswere combined and concentrated to dryness to yield Compound 105a(quantitative). The structure was confirmed by LCMS.

Compound 113 (1.25 g, 2.7 mmol), HBTU (3.2 g, 8.4 mmol) and DIEA (2.8mL, 16.2 mmol) were dissolved in anhydrous DMF (17 mL) and the reactionmixture was stirred at room temperature for 5 min. To this a solution ofCompound 105a (3.77 g, 8.4 mmol) in anhydrous DMF (20 mL) was added. Thereaction was stirred at room temperature for 6 h. Solvent was removedunder reduced pressure to get an oil. The residue was dissolved inCH₂Cl₂ (100 mL) and washed with aqueous saturated NaHCO₃ solution (100mL) and brine (100 mL). The organic phase was separated, dried (Na₂SO₄),filtered and evaporated. The residue was purified by silica gel columnchromatography and eluted with 10 to 20% MeOH in dichloromethane toyield Compound 114 (1.45 g, 30%). The structure was confirmed by LCMSand ¹H NMR analysis.

Compound 114 (1.43 g, 0.8 mmol) was dissolved in 1:1 methanol/ethylacetate (4 mL/4 mL). Palladium on carbon (wet, 0.14 g) was added. Thereaction mixture was flushed with hydrogen and stirred at roomtemperature under hydrogen for 12 h. The reaction mixture was filteredthrough a pad of celite. The celite pad was washed with methanol/ethylacetate (1:1). The filtrate and the washings were combined together andevaporated under reduced pressure to yield Compound 115 (quantitative).The structure was confirmed by LCMS and ¹H NMR analysis.

Compound 83a (0.17 g, 0.75 mmol), HBTU (0.31 g, 0.83 mmol) and DIEA(0.26 mL, 1.5 mmol) were dissolved in anhydrous DMF (5 mL) and thereaction mixture was stirred at room temperature for 5 min. To this asolution of Compound 115 (1.22 g, 0.75 mmol) in anhydrous DMF was addedand the reaction was stirred at room temperature for 6 h. The solventwas removed under reduced pressure and the residue was dissolved inCH₂Cl₂. The organic layer was washed aqueous saturated NaHCO₃ solutionand brine and dried over anhydrous Na₂SO₄ and filtered. The organiclayer was concentrated to dryness and the residue obtained was purifiedby silica gel column chromatography and eluted with 3 to 15% MeOH indichloromethane to yield Compound 116 (0.84 g, 61%). The structure wasconfirmed by LC MS and ¹H NMR analysis.

Compound 116 (0.74 g, 0.4 mmol) was dissolved in 1:1 methanol/ethylacetate (5 mL/5 mL). Palladium on carbon (wet, 0.074 g) was added. Thereaction mixture was flushed with hydrogen and stirred at roomtemperature under hydrogen for 12 h. The reaction mixture was filteredthrough a pad of celite. The celite pad was washed with methanol/ethylacetate (1:1). The filtrate and the washings were combined together andevaporated under reduced pressure to yield compound 117 (0.73 g, 98%).The structure was confirmed by LCMS and ¹H NMR analysis.

Compound 117 (0.63 g, 0.36 mmol) was dissolved in anhydrous DMF (3 mL).To this solution N,N-Diisopropylethylamine (70 μL, 0.4 mmol) andpentafluorophenyl trifluoroacetate (72 μL, 0.42 mmol) were added. Thereaction mixture was stirred at room temperature for 12 h and pouredinto a aqueous saturated NaHCO₃ solution. The mixture was extracted withdichloromethane, washed with brine and dried over anhydrous Na₂SO₄. Thedichloromethane solution was concentrated to dryness and purified withsilica gel column chromatography and eluted with 5 to 10% MeOH indichloromethane to yield compound 118 (0.51 g, 79%). The structure wasconfirmed by LCMS and ¹H and ¹H and ¹⁹F NMR.

Oligomeric Compound 119, comprising a GalNAc₃-7 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-7 (GalNAc₃-7_(a))can be combined with any cleavable moiety to provide a variety ofconjugate groups. In certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-7 (GalNAc₃-7_(a)-CM-) is shown below:

Example 49: Preparation of Oligonucleotide 132 Comprising GalNAc₃-5

Compound 120 (14.01 g, 40 mmol) and HBTU (14.06 g, 37 mmol) weredissolved in anhydrous DMF (80 mL). Triethylamine (11.2 mL, 80.35 mmol)was added and stirred for 5 min. The reaction mixture was cooled in anice bath and a solution of compound 121 (10 g, mmol) in anhydrous DMF(20 mL) was added. Additional triethylamine (4.5 mL, 32.28 mmol) wasadded and the reaction mixture was stirred for 18 h under an argonatmosphere. The reaction was monitored by TLC (ethyl acetate:hexane;1:1; Rf=0.47). The solvent was removed under reduced pressure. Theresidue was taken up in EtOAc (300 mL) and washed with 1M NaHSO₄ (3×150mL), aqueous saturated NaHCO₃ solution (3×150 mL) and brine (2×100 mL).Organic layer was dried with Na₂SO₄. Drying agent was removed byfiltration and organic layer was concentrated by rotary evaporation.Crude mixture was purified by silica gel column chromatography andeluted by using 35-50% EtOAc in hexane to yield a compound 122 (15.50 g,78.13%). The structure was confirmed by LCMS and ¹H NMR analysis. Massm/z 589.3 [M+H]⁺.

A solution of LiOH (92.15 mmol) in water (20 mL) and THF (10 mL) wasadded to a cooled solution of Compound 122 (7.75 g, 13.16 mmol)dissolved in methanol (15 mL). The reaction mixture was stirred at roomtemperature for 45 min. and monitored by TLC (EtOAc:hexane; 1:1). Thereaction mixture was concentrated to half the volume under reducedpressure. The remaining solution was cooled an ice bath and neutralizedby adding concentrated HCl. The reaction mixture was diluted, extractedwith EtOAc (120 mL) and washed with brine (100 mL). An emulsion formedand cleared upon standing overnight. The organic layer was separateddried (Na₂SO₄), filtered and evaporated to yield Compound 123 (8.42 g).Residual salt is the likely cause of excess mass. LCMS is consistentwith structure. Product was used without any further purification.M.W.cal: 574.36; M.W.fd: 575.3[M+H]⁺.

Compound 126 was synthesized following the procedure described in theliterature (J. Am. Chem. Soc. 2011, 133, 958-963).

Compound 123 (7.419 g, 12.91 mmol), HOBt (3.49 g, 25.82 mmol) andcompound 126 (6.33 g, 16.14 mmol) were dissolved in and DMF (40 mL) andthe resulting reaction mixture was cooled in an ice bath. To thisN,N-Diisopropylethylamine (4.42 mL, 25.82 mmol), PyBop (8.7 g, 16.7mmol) followed by Bop coupling reagent (1.17 g, 2.66 mmol) were addedunder an argon atmosphere. The ice bath was removed and the solution wasallowed to warm to room temperature. The reaction was completed after 1h as determined by TLC (DCM:MeOH:AA; 89:10:1). The reaction mixture wasconcentrated under reduced pressure. The residue was dissolved in EtOAc(200 mL) and washed with 1 M NaHSO₄ (3×100 mL), aqueous saturated NaHCO₃(3×100 mL) and brine (2×100 mL). The organic phase separated dried(Na₂SO₄), filtered and concentrated. The residue was purified by silicagel column chromatography with a gradient of 50% hexanes/EtOAC to 100%EtOAc to yield Compound 127 (9.4 g) as a white foam. LCMS and ¹H NMRwere consistent with structure. Mass m/z 778.4 [M+H]⁺.

Trifluoroacetic acid (12 mL) was added to a solution of compound 127(1.57 g, 2.02 mmol) in dichloromethane (12 mL) and stirred at roomtemperature for 1 h. The reaction mixture was co-evaporated with toluene(30 mL) under reduced pressure to dryness. The residue obtained wasco-evaporated twice with acetonitrile (30 mL) and toluene (40 mL) toyield Compound 128 (1.67 g) as trifluoro acetate salt and used for nextstep without further purification. LCMS and ¹H NMR were consistent withstructure. Mass m/z 478.2 [M+H]⁺.

Compound 7 (0.43 g, 0.963 mmol), HATU (0.35 g, 0.91 mmol), and HOAt(0.035 g, 0.26 mmol) were combined together and dried for 4 h over P₂O₅under reduced pressure in a round bottom flask and then dissolved inanhydrous DMF (1 mL) and stirred for 5 min. To this a solution ofcompound 128 (0.20 g, 0.26 mmol) in anhydrous DMF (0.2 mL) andN,N-Diisopropylethylamine (0.2 mL) was added. The reaction mixture wasstirred at room temperature under an argon atmosphere. The reaction wascomplete after 30 min as determined by LCMS and TLC (7% MeOH/DCM). Thereaction mixture was concentrated under reduced pressure. The residuewas dissolved in DCM (30 mL) and washed with 1 M NaHSO₄ (3×20 mL),aqueous saturated NaHCO₃ (3×20 mL) and brine (3×20 mL). The organicphase was separated, dried over Na₂SO₄, filtered and concentrated. Theresidue was purified by silica gel column chromatography using 5-15%MeOH in dichloromethane to yield Compound 129 (96.6 mg). LC MS and ¹HNMR are consistent with structure. Mass m/z 883.4 [M+2H]⁺.

Compound 129 (0.09 g, 0.051 mmol) was dissolved in methanol (5 mL) in 20mL scintillation vial. To this was added a small amount of 10% Pd/C(0.015 mg) and the reaction vessel was flushed with H₂ gas. The reactionmixture was stirred at room temperature under H₂ atmosphere for 18 h.The reaction mixture was filtered through a pad of Celite and the Celitepad was washed with methanol. The filtrate washings were pooled togetherand concentrated under reduced pressure to yield Compound 130 (0.08 g).LCMS and NMR were consistent with structure. The product was usedwithout further purification. Mass m/z 838.3 [M+2H]⁺.

To a 10 mL pointed round bottom flask were added compound 130 (75.8 mg,0.046 mmol), 0.37 M pyridine/DMF (200 μL) and a stir bar. To thissolution was added 0.7 M pentafluorophenyl trifluoroacetate/DMF (100 μL)drop wise with stirring. The reaction was completed after 1 h asdetermined by LC MS. The solvent was removed under reduced pressure andthe residue was dissolved in CHCl₃ (˜10 mL). The organic layer waspartitioned against NaHSO₄ (1 M, 10 mL), aqueous saturated NaHCO₃ (10mL) and brine (10 mL) three times each. The organic phase separated anddried over Na₂SO₄, filtered and concentrated to yield Compound 131 (77.7mg). LCMS is consistent with structure. Used without furtherpurification. Mass m/z 921.3 [M+2H]⁺.

Oligomeric Compound 132, comprising a GalNAc₃-5 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-5 (GalNAc₃-5_(a))can be combined with any cleavable moiety to provide a variety ofconjugate groups. In certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-5 (GalNAc₃-5_(a)-CM-) is shown below:

Example 50: Preparation of Oligonucleotide 144 Comprising GalNAc₄-11

Synthesis of Compound 134. To a Merrifield flask was added aminomethylVIMAD resin (2.5 g, 450 μmol/g) that was washed with acetonitrile,dimethylformamide, dichloromethane and acetonitrile. The resin wasswelled in acetonitrile (4 mL). Compound 133 was pre-activated in a 100mL round bottom flask by adding 20 (1.0 mmol, 0.747 g), TBTU (1.0 mmol,0.321 g), acetonitrile (5 mL) and DIEA (3.0 mmol, 0.5 mL). This solutionwas allowed to stir for 5 min and was then added to the Merrifield flaskwith shaking. The suspension was allowed to shake for 3 h. The reactionmixture was drained and the resin was washed with acetonitrile, DMF andDCM. New resin loading was quantitated by measuring the absorbance ofthe DMT cation at 500 nm (extinction coefficient=76000) in DCM anddetermined to be 238 μmol/g. The resin was capped by suspending in anacetic anhydride solution for ten minutes three times.

The solid support bound compound 141 was synthesized using iterativeFmoc-based solid phase peptide synthesis methods. A small amount ofsolid support was withdrawn and suspended in aqueous ammonia (28-30 wt%) for 6 h. The cleaved compound was analyzed by LC-MS and the observedmass was consistent with structure. Mass m/z 1063.8 [M+2H]⁺.

The solid support bound compound 142 was synthesized using solid phasepeptide synthesis methods.

The solid support bound compound 143 was synthesized using standardsolid phase synthesis on a DNA synthesizer.

The solid support bound compound 143 was suspended in aqueous ammonia(28-30 wt %) and heated at 55° C. for 16 h. The solution was cooled andthe solid support was filtered. The filtrate was concentrated and theresidue dissolved in water and purified by HPLC on a strong anionexchange column. The fractions containing full length compound 144 werepooled together and desalted. The resulting GalNAc₄-11 conjugatedoligomeric compound was analyzed by LC-MS and the observed mass wasconsistent with structure.

The GalNAc4 cluster portion of the conjugate group GalNAc₄-11(GalNAc₄-11_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. In certain embodiments, the cleavablemoiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₄-11 (GalNAc₄-11_(a)-CM) is shown below:

Example 51: Preparation of Oligonucleotide 155 Comprising GalNAc₃-6

Compound 146 was synthesized as described in the literature (AnalyticalBiochemistry 1995, 229, 54-60).

Compound 4 (15 g, 45.55 mmol) and compound 35b (14.3 grams, 57 mmol)were dissolved in CH₂Cl₂ (200 ml). Activated molecular sieves (4 Å. 2 g,powdered) were added, and the reaction was allowed to stir for 30minutes under nitrogen atmosphere. TMS-OTf was added (4.1 ml, 22.77mmol) and the reaction was allowed to stir at room temp overnight. Uponcompletion, the reaction was quenched by pouring into solution ofsaturated aqueous NaHCO₃ (500 ml) and crushed ice (˜150 g). The organiclayer was separated, washed with brine, dried over MgSO₄, filtered, andwas concentrated to an orange oil under reduced pressure. The crudematerial was purified by silica gel column chromatography and elutedwith 2-10% MeOH in CH₂Cl₂ to yield Compound 112 (16.53 g, 63%). LCMS and¹H NMR were consistent with the expected compound.

Compound 112 (4.27 g, 7.35 mmol) was dissolved in 1:1 MeOH/EtOAc (40ml). The reaction mixture was purged by bubbling a stream of argonthrough the solution for 15 minutes. Pearlman's catalyst (palladiumhydroxide on carbon, 400 mg) was added, and hydrogen gas was bubbledthrough the solution for 30 minutes. Upon completion (TLC 10% MeOH inCH₂Cl₂, and LCMS), the catalyst was removed by filtration through a padof celite. The filtrate was concentrated by rotary evaporation, and wasdried briefly under high vacuum to yield Compound 105a (3.28 g). LCMSand 1H NMR were consistent with desired product.

Compound 147 (2.31 g, 11 mmol) was dissolved in anhydrous DMF (100 mL).N,N-Diisopropylethylamine (DIEA, 3.9 mL, 22 mmol) was added, followed byHBTU (4 g, 10.5 mmol). The reaction mixture was allowed to stir for ˜15minutes under nitrogen. To this a solution of compound 105a (3.3 g, 7.4mmol) in dry DMF was added and stirred for 2 h under nitrogenatmosphere. The reaction was diluted with EtOAc and washed withsaturated aqueous NaHCO₃ and brine. The organics phase was separated,dried (MgSO₄), filtered, and concentrated to an orange syrup. The crudematerial was purified by column chromatography 2-5% MeOH in CH₂Cl₂ toyield Compound 148 (3.44 g, 73%). LCMS and ¹H NMR were consistent withthe expected product.

Compound 148 (3.3 g, 5.2 mmol) was dissolved in 1:1 MeOH/EtOAc (75 ml).The reaction mixture was purged by bubbling a stream of argon throughthe solution for 15 minutes. Pearlman's catalyst (palladium hydroxide oncarbon) was added (350 mg). Hydrogen gas was bubbled through thesolution for 30 minutes. Upon completion (TLC 10% MeOH in DCM, andLCMS), the catalyst was removed by filtration through a pad of celite.The filtrate was concentrated by rotary evaporation, and was driedbriefly under high vacuum to yield Compound 149 (2.6 g). LCMS wasconsistent with desired product. The residue was dissolved in dry DMF(10 ml) was used immediately in the next step.

Compound 146 (0.68 g, 1.73 mmol) was dissolved in dry DMF (20 ml). Tothis DIEA (450 μL, 2.6 mmol, 1.5 eq.) and HBTU (1.96 g, 0.5.2 mmol) wereadded. The reaction mixture was allowed to stir for 15 minutes at roomtemperature under nitrogen. A solution of compound 149 (2.6 g) inanhydrous DMF (10 mL) was added. The pH of the reaction was adjusted topH=9-10 by addition of DIEA (if necessary). The reaction was allowed tostir at room temperature under nitrogen for 2 h. Upon completion thereaction was diluted with EtOAc (100 mL), and washed with aqueoussaturated aqueous NaHCO₃, followed by brine. The organic phase wasseparated, dried over MgSO₄, filtered, and concentrated. The residue waspurified by silica gel column chromatography and eluted with 2-10% MeOHin CH₂Cl₂ to yield Compound 150 (0.62 g, 20%). LCMS and ¹H NMR wereconsistent with the desired product.

Compound 150 (0.62 g) was dissolved in 1:1 MeOH/EtOAc (5 L). Thereaction mixture was purged by bubbling a stream of argon through thesolution for 15 minutes. Pearlman's catalyst (palladium hydroxide oncarbon) was added (60 mg). Hydrogen gas was bubbled through the solutionfor 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), thecatalyst was removed by filtration (syringe-tip Teflon filter, 0.45 μm).The filtrate was concentrated by rotary evaporation, and was driedbriefly under high vacuum to yield Compound 151 (0.57 g). The LCMS wasconsistent with the desired product. The product was dissolved in 4 mLdry DMF and was used immediately in the next step.

Compound 83a (0.11 g, 0.33 mmol) was dissolved in anhydrous DMF (5 mL)and N,N-Diisopropylethylamine (75 μL, 1 mmol) and PFP-TFA (90 μL, 0.76mmol) were added. The reaction mixture turned magenta upon contact, andgradually turned orange over the next 30 minutes. Progress of reactionwas monitored by TLC and LCMS. Upon completion (formation of the PFPester), a solution of compound 151 (0.57 g, 0.33 mmol) in DMF was added.The pH of the reaction was adjusted to pH=9-10 by addition ofN,N-Diisopropylethylamine (if necessary). The reaction mixture wasstirred under nitrogen for ˜30 min. Upon completion, the majority of thesolvent was removed under reduced pressure. The residue was diluted withCH₂Cl₂ and washed with aqueous saturated NaHCO₃, followed by brine. Theorganic phase separated, dried over MgSO₄, filtered, and concentrated toan orange syrup. The residue was purified by silica gel columnchromatography (2-10% MeOH in CH₂Cl₂) to yield Compound 152 (0.35 g,55%). LCMS and ¹H NMR were consistent with the desired product.

Compound 152 (0.35 g, 0.182 mmol) was dissolved in 1:1 MeOH/EtOAc (10mL). The reaction mixture was purged by bubbling a stream of argon thruthe solution for 15 minutes. Pearlman's catalyst (palladium hydroxide oncarbon) was added (35 mg). Hydrogen gas was bubbled thru the solutionfor 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), thecatalyst was removed by filtration (syringe-tip Teflon filter, 0.45 μm).The filtrate was concentrated by rotary evaporation, and was driedbriefly under high vacuum to yield Compound 153 (0.33 g, quantitative).The LCMS was consistent with desired product.

Compound 153 (0.33 g, 0.18 mmol) was dissolved in anhydrous DMF (5 mL)with stirring under nitrogen. To this N,N-Diisopropylethylamine (65 μL,0.37 mmol) and PFP-TFA (35 μL, 0.28 mmol) were added. The reactionmixture was stirred under nitrogen for ˜30 min. The reaction mixtureturned magenta upon contact, and gradually turned orange. The pH of thereaction mixture was maintained at pH=9-10 by adding moreN,-Diisopropylethylamine. The progress of the reaction was monitored byTLC and LCMS. Upon completion, the majority of the solvent was removedunder reduced pressure. The residue was diluted with CH₂Cl₂ (50 mL), andwashed with saturated aqueous NaHCO₃, followed by brine. The organiclayer was dried over MgSO₄, filtered, and concentrated to an orangesyrup. The residue was purified by column chromatography and eluted with2-10% MeOH in CH₂Cl₂ to yield Compound 154 (0.29 g, 79%). LCMS and ¹HNMR were consistent with the desired product.

Oligomeric Compound 155, comprising a GalNAc₃-6 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-6 (GalNAc₃-6_(a))can be combined with any cleavable moiety to provide a variety ofconjugate groups. In certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—.

The structure of GalNAc₃-6 (GalNAc₃-6_(a)-CM-) is shown below:

Example 52: Preparation of Oligonucleotide 160 Comprising GalNAc₃-9

Compound 156 was synthesized following the procedure described in theliterature (J. Med. Chem. 2004, 47, 5798-5808).

Compound 156, (18.60 g, 29.28 mmol) was dissolved in methanol (200 mL).Palladium on carbon (6.15 g, 10 wt %, loading (dry basis), matrix carbonpowder, wet) was added. The reaction mixture was stirred at roomtemperature under hydrogen for 18 h. The reaction mixture was filteredthrough a pad of celite and the celite pad was washed thoroughly withmethanol. The combined filtrate was washed and concentrated to dryness.The residue was purified by silica gel column chromatography and elutedwith 5-10% methanol in dichloromethane to yield Compound 157 (14.26 g,89%). Mass m/z 544.1 [M−H]⁻.

Compound 157 (5 g, 9.17 mmol) was dissolved in anhydrous DMF (30 mL).HBTU (3.65 g, 9.61 mmol) and N,N-Diisopropylethylamine (13.73 mL, 78.81mmol) were added and the reaction mixture was stirred at roomtemperature for 5 minutes. To this a solution of compound 47 (2.96 g,7.04 mmol) was added. The reaction was stirred at room temperature for 8h. The reaction mixture was poured into a saturated NaHCO₃ aqueoussolution. The mixture was extracted with ethyl acetate and the organiclayer was washed with brine and dried (Na₂SO₄), filtered and evaporated.The residue obtained was purified by silica gel column chromatographyand eluted with 50% ethyl acetate in hexane to yield compound 158 (8.25g, 73.3%). The structure was confirmed by MS and ¹H NMR analysis.

Compound 158 (7.2 g, 7.61 mmol) was dried over P₂O₅ under reducedpressure. The dried compound was dissolved in anhydrous DMF (50 mL). Tothis 1H tetrazole (0.43 g, 6.09 mmol) and N-methylimidazole (0.3 mL,3.81 mmol) and 2-cyanoethyl-N,N,N′,N′-tetraisopropyl phosphorodiamidite(3.65 mL, 11.50 mmol) were added. The reaction mixture was stirred tunder an argon atmosphere for 4 h. The reaction mixture was diluted withethyl acetate (200 mL). The reaction mixture was washed with saturatedNaHCO₃ and brine. The organic phase was separated, dried (Na₂SO₄),filtered and evaporated. The residue was purified by silica gel columnchromatography and eluted with 50-90% ethyl acetate in hexane to yieldCompound 159 (7.82 g, 80.5%). The structure was confirmed by LCMS and³¹P NMR analysis.

Oligomeric Compound 160, comprising a GalNAc₃-9 conjugate group, wasprepared using standard oligonucleotide synthesis procedures. Threeunits of compound 159 were coupled to the solid support, followed bynucleotide phosphoramidites. Treatment of the protected oligomericcompound with aqueous ammonia yielded compound 160. The GalNAc₃ clusterportion of the conjugate group GalNAc₃-9 (GalNAc₃-9_(a)) can be combinedwith any cleavable moiety to provide a variety of conjugate groups. Incertain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-9(GalNAc₃-9_(a)-CM) is shown below:

Example 53: Alternate Procedure for Preparation of Compound 18(GalNAc₃-1a and GalNAc₃-3a)

Lactone 161 was reacted with diamino propane β-5 eq) or Mono-Bocprotected diamino propane (1 eq) to provide alcohol 162a or 162b. Whenunprotected propanediamine was used for the above reaction, the excessdiamine was removed by evaporation under high vacuum and the free aminogroup in 162a was protected using CbzCl to provide 162b as a white solidafter purification by column chromatography. Alcohol 162b was furtherreacted with compound 4 in the presence of TMSOTf to provide 163a whichwas converted to 163b by removal of the Cbz group using catalytichydrogenation. The pentafluorophenyl (PFP) ester 164 was prepared byreacting triacid 113 (see Example 48) with PFPTFA (3.5 eq) and pyridine(3.5 eq) in DMF (0.1 to 0.5 M). The triester 164 was directly reactedwith the amine 163b (3-4 eq) and DIPEA (3-4 eq) to provide Compound 18.The above method greatly facilitates purification of intermediates andminimizes the formation of byproducts which are formed using theprocedure described in Example 4.

Example 54: Alternate Procedure for Preparation of Compound 18(GalNAc₃-1a and GalNAc₃-3a)

The triPFP ester 164 was prepared from acid 113 using the procedureoutlined in example 53 above and reacted with mono-Boc protected diamineto provide 165 in essentially quantitative yield. The Boc groups wereremoved with hydrochloric acid or trifluoroacetic acid to provide thetriamine which was reacted with the PFP activated acid 166 in thepresence of a suitable base such as DIPEA to provide Compound 18.

The PFP protected Gal-NAc acid 166 was prepared from the correspondingacid by treatment with PFPTFA (1-1.2 eq) and pyridine (1-1.2 eq) in DMF.The precursor acid in turn was prepared from the corresponding alcoholby oxidation using TEMPO (0.2 eq) and BAIB in acetonitrile and water.The precursor alcohol was prepared from sugar intermediate 4 by reactionwith 1,6-hexanediol (or 1,5-pentanediol or other diol for other nvalues) (2-4 eq) and TMSOTf using conditions described previously inexample 47.

Example 55: Dose-Dependent Study of Oligonucleotides Comprising Either a3′ or 5′-Conjugate Group (Comparison of GalNAc₃-1, 3, 8 and 9) TargetingSRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 wasincluded as a standard. Each of the various GalNAc₃ conjugate groups wasattached at either the 3′ or 5′ terminus of the respectiveoligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside(cleavable moiety).

TABLE 39 Modified ASO targeting SRB-1 SEQ ID ASO Sequence (5′ to 3′)Motif Conjugate No. ISIS 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5none 252 (parent) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 655861 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5GalNAc ₃ -1 253 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo)A _(do′) -GalNAc ₃ -1 _(a) ISIS 664078 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5GalNAc ₃ -9 253 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo)A _(do′) -GalNAc ₃ -9 _(a) ISIS 661161 GalNAc ₃ -3 _(a) - _(o′) A _(do)5/10/5 GalNAc ₃ -3 254 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 665001GalNAc ₃ -8 _(a) - _(o′) A _(do) 5/10/5 GalNAc ₃ -8 254 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. Thestructure of GalNAc₃-9 was shown previously in Example 52. The structureof GalNAc₃-3 was shown previously in Example 39. The structure ofGalNAc₃-8 was shown previously in Example 47.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 353382,655861, 664078, 661161, 665001 or with saline. Each treatment groupconsisted of 4 animals. The mice were sacrificed 72 hours following thefinal administration to determine the liver SRB-1 mRNA levels usingreal-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. The resultsbelow are presented as the average percent of SRB-1 mRNA levels for eachtreatment group, normalized to the saline control.

As illustrated in Table 40, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, theantisense oligonucleotides comprising the phosphodiester linkedGalNAc₃-1 and GalNAc₃-9 conjugates at the 3′ terminus (ISIS 655861 andISIS 664078) and the GalNAc₃-3 and GalNAc₃-8 conjugates linked at the 5′terminus (ISIS 661161 and ISIS 665001) showed substantial improvement inpotency compared to the unconjugated antisense oligonucleotide (ISIS353382). Furthermore, ISIS 664078, comprising a GalNAc₃-9 conjugate atthe 3′ terminus was essentially equipotent compared to ISIS 655861,which comprises a GalNAc₃-1 conjugate at the 3′ terminus. The 5′conjugated antisense oligonucleotides, ISIS 661161 and ISIS 665001,comprising a GalNAc₃-3 or GalNAc₃-9, respectively, had increased potencycompared to the 3′ conjugated antisense oligonucleotides (ISIS 655861and ISIS 664078).

TABLE 40 ASOs containing GalNAc₃-1, 3, 8 or 9 targeting SRB-1 DosageSRB-1 mRNA ISIS No. (mg/kg) (% Saline) Conjugate Saline n/a 100 353382 388 none 10 68 30 36 655861 0.5 98 GalNac₃-1 (3′) 1.5 76 5 31 15 20664078 0.5 88 GalNac₃-9 (3′) 1.5 85 5 46 15 20 661161 0.5 92 GalNac₃-3(5′) 1.5 59 5 19 15 11 665001 0.5 100 GalNac₃-8 (5′) 1.5 73 5 29 15 13

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The change in body weights was evaluated with nosignificant change from the saline group. ALTs, ASTs, total bilirubinand BUN values are shown in the table below.

TABLE 41 ISIS Dosage Total No. mg/kg ALT AST Bilirubin BUN ConjugateSaline 24 59 0.1 37.52 353382 3 21 66 0.2 34.65 none 10 22 54 0.2 34.230 22 49 0.2 33.72 655861 0.5 25 62 0.2 30.65 GalNac₃-1 (3′) 1.5 23 480.2 30.97 5 28 49 0.1 32.92 15 40 97 0.1 31.62 664078 0.5 40 74 0.1 35.3GalNac₃-9 (3′) 1.5 47 104 0.1 32.75 5 20 43 0.1 30.62 15 38 92 0.1 26.2661161 0.5 101 162 0.1 34.17 GalNac₃-3 (5′) 1.5 g 42 100 0.1 33.37   5 g23 99 0.1 34.97 15 53 83 0.1 34.8 665001 0.5 28 54 0.1 31.32 GalNac₃-8(5′) 1.5 42 75 0.1 32.32 5 24 42 0.1 31.85 15 32 67 0.1 31.

Example 56: Dose-Dependent Study of Oligonucleotides Comprising Either a3′ or 5′-Conjugate Group (Comparison of GalNAc₃-1, 2, 3, 5, 6, 7 and 10)Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 wasincluded as a standard. Each of the various GalNAc₃ conjugate groups wasattached at the 5′ terminus of the respective oligonucleotide by aphosphodiester linked 2′-deoxyadenosine nucleoside (cleavable moiety)except for ISIS 655861 which had the GalNAc₃ conjugate group attached atthe 3′ terminus.

TABLE 42 Modified ASO targeting SRB-1 SEQ ID ASO Sequence (5′ to 3′)Motif Conjugate No. ISIS 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5no conjugate 252 (parent) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 655861 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5GalNAc ₃ -1 253 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo)A _(do′) -GalNAc ₃ -1 _(a) ISIS 664507 GalNAc ₃ -2 _(a) - _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5GalNAc ₃ -2 254 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 661161GalNAc ₃ -3 _(a) - _(o′) A _(do) 5/10/5 GalNAc ₃ -3 254 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666224 GalNAc ₃ -5 _(a) - _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -5254 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666961 GalNAc ₃ -6 _(a) - _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -6254 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666981 GalNAc ₃ -7 _(a) - _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -7254 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666881 GalNAc ₃ -10 _(a) - _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5GalNAc ₃ -10 254 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. Thestructure of GalNAc₃-2_(a) was shown previously in Example 37. Thestructure of GalNAc₃-3_(a) was shown previously in Example 39. Thestructure of GalNAc₃-5_(a) was shown previously in Example 49. Thestructure of GalNAc₃-6_(a) was shown previously in Example 51. Thestructure of GalNAc₃-7_(a) was shown previously in Example 48. Thestructure of GalNAc₃-10_(a) was shown previously in Example 46.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 353382,655861, 664507, 661161, 666224, 666961, 666981, 666881 or with saline.Each treatment group consisted of 4 animals. The mice were sacrificed 72hours following the final administration to determine the liver SRB-1mRNA levels using real-time PCR and RIBOGREEN® RNA quantificationreagent (Molecular Probes, Inc. Eugene, Oreg.) according to standardprotocols. The results below are presented as the average percent ofSRB-1 mRNA levels for each treatment group, normalized to the salinecontrol.

As illustrated in Table 43, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, theconjugated antisense oligonucleotides showed substantial improvement inpotency compared to the unconjugated antisense oligonucleotide (ISIS353382). The 5′ conjugated antisense oligonucleotides showed a slightincrease in potency compared to the 3′ conjugated antisenseoligonucleotide.

TABLE 43 Dosage SRB-1 mRNA ISIS No. (mg/kg) (% Saline) Conjugate Salinen/a 100.0 353382 3 96.0 none 10 73.1 30 36.1 655861 0.5 99.4 GalNac₃-1(3′)  1.5 81.2 5 33.9 15 15.2 664507 0.5 102.0 GalNac₃-2 (5′)  1.5 73.25 31.3 15 10.8 661161 0.5 90.7 GalNac₃-3 (5′)  1.5 67.6 5 24.3 15 11.5666224 0.5 96.1 GalNac₃-5 (5′)  1.5 61.6 5 25.6 15 11.7 666961 0.5 85.5GalNAc₃-6 (5′)  1.5 56.3 5 34.2 15 13.1 666981 0.5 84.7 GalNAc₃-7 (5′) 1.5 59.9 5 24.9 15 8.5 666881 0.5 100.0 GalNAc₃-10 (5′) 1.5 65.8 5 26.015 13.0

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The change in body weights was evaluated with nosignificant change from the saline group. ALTs, ASTs, total bilirubinand BUN values are shown in Table 44 below.

TABLE 44 ISIS Dosage Total No. mg/kg ALT AST Bilirubin BUN ConjugateSaline 26 57 0.2 27 353382 3 25 92 0.2 27 none 10 23 40 0.2 25 30 29 540.1 28 655861 0.5 25 71 0.2 34 GalNac₃-1 (3′) 1.5 28 60 0.2 26 5 26 630.2 28 15 25 61 0.2 28 664507 0.5 25 62 0.2 25 GalNac₃-2 (5′) 1.5 24 490.2 26 5 21 50 0.2 26 15 59 84 0.1 22 661161 0.5 20 42 0.2 29 GalNac₃-3(5′) 1.5 g 37 74 0.2 25   5 g 28 61 0.2 29 15 21 41 0.2 25 666224 0.5 3448 0.2 21 GalNac₃-5 (5′) 1.5 23 46 0.2 26 5 24 47 0.2 23 15 32 49 0.1 26666961 0.5 17 63 0.2 26 GalNAc₃-6 (5′) 1.5 23 68 0.2 26 5 25 66 0.2 2615 29 107 0.2 28 666981 0.5 24 48 0.2 26 GalNAc₃-7 (5′) 1.5 30 55 0.2 245 46 74 0.1 24 15 29 58 0.1 26 666881 0.5 20 65 0.2 27 GalNAc₃-10 (5′)1.5 23 59 0.2 24 5 45 70 0.2 26 15 21 57 0.2 24

Example 57: Duration of Action Study of Oligonucleotides Comprising a3′-Conjugate Group Targeting ApoC III In Vivo

Mice were injected once with the doses indicated below and monitoredover the course of 42 days for ApoC-III and plasma triglycerides (PlasmaTG) levels. The study was performed using 3 transgenic mice that expresshuman APOC-III in each group.

TABLE 45 Modified ASO targeting ApoC III SEQ ID ASO Sequence (5′ to 3′)Linkages No. ISIS A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) PS 244 304801 ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) ISISA_(es)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds)^(m)C_(ds) ^(m)C_(ds) PS 245 647535 A_(ds)G_(ds)^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a) ISISA_(es)G_(eo) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds)^(m)C_(ds) ^(m)C_(ds) PO/PS 245 647536 A_(ds)G_(ds)^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a)

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9.

TABLE 46 ApoC III mRNA (% Saline on Day 1) and Plasma TG Levels (%Saline on Day 1) Day Day Day Day Day ASO Dose Target 3 7 14 35 42 Saline 0 mg/kg ApoC-III 98 100 100 95 116 ISIS 304801 30 mg/kg ApoC-III 28 3041 65 74 ISIS 647535 10 mg/kg ApoC-III 16 19 25 74 94 ISIS 647536 10mg/kg ApoC-III 18 16 17 35 51 Saline  0 mg/kg Plasma TG 121 130 123 105109 ISIS 304801 30 mg/kg Plasma TG 34 37 50 69 69 ISIS 647535 10 mg/kgPlasma TG 18 14 24 18 71 ISIS 647536 10 mg/kg Plasma TG 21 19 15 32 35

As can be seen in the table above the duration of action increased withaddition of the 3′-conjugate group compared to the unconjugatedoligonucleotide. There was a further increase in the duration of actionfor the conjugated mixed PO/PS oligonucleotide 647536 as compared to theconjugated full PS oligonucleotide 647535.

Example 58: Dose-Dependent Study of Oligonucleotides Comprising a3′-Conjugate Group (Comparison of GalNAc₃-1 and GalNAc₄-11) TargetingSRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Unconjugated ISIS 440762 wasincluded as an unconjugated standard. Each of the conjugate groups wereattached at the 3′ terminus of the respective oligonucleotide by aphosphodiester linked 2′-deoxyadenosine nucleoside cleavable moiety.

The structure of GalNAc₃-1_(a) was shown previously in Example 9. Thestructure of GalNAc₃-11_(a) was shown previously in Example 50.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously once at the dosage shown below with ISIS 440762,651900, 663748 or with saline. Each treatment group consisted of 4animals. The mice were sacrificed 72 hours following the finaladministration to determine the liver SRB-1 mRNA levels using real-timePCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc.Eugene, Oreg.) according to standard protocols. The results below arepresented as the average percent of SRB-1 mRNA levels for each treatmentgroup, normalized to the saline control.

As illustrated in Table 47, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. The antisenseoligonucleotides comprising the phosphodiester linked GalNAc₃-1 andGalNAc₄-11 conjugates at the 3′ terminus (ISIS 651900 and ISIS 663748)showed substantial improvement in potency compared to the unconjugatedantisense oligonucleotide (ISIS 440762). The two conjugatedoligonucleotides, GalNAc₃-1 and GalNAc₄-11, were equipotent.

TABLE 47 Modified ASO targeting SRB-1 % Saline SEQ ID ASO Sequence(5′ to 3′) Dose mg/kg control No. Saline 100 ISIS 440762 T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 0.673.45 246 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) 2 59.66 6 23.50 ISIS 651900T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)0.2 62.75 247 ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ko)A_(do′)-GalNAc ₃ -1 _(a)0.6 29.14 2 8.61 6 5.62 ISIS 663748 T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 0.2 63.99 247 ^(m)C_(ds)T_(ds)T_(ks)^(m)C_(ko)A_(do′)-GalNAc ₄ -11 _(a) 0.6 33.53 2 7.58 6 5.52

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside; “d”indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioateinternucleoside linkage (PS); “o” indicates a phosphodiesterinternucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—.Conjugate groups are in bold.

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The change in body weights was evaluated with nosignificant change from the saline group. ALTs, ASTs, total bilirubinand BUN values are shown in Table 48 below.

TABLE 48 ISIS Dosage Total No. mg/kg ALT AST Bilirubin BUN ConjugateSaline 30 76 0.2 40 440762 0.60 32 70 0.1 35 none 2 26 57 0.1 35 6 31 480.1 39 651900 0.2 32 115 0.2 39 GalNac₃-1 (3′) 0.6 33 61 0.1 35 2 30 500.1 37 6 34 52 0.1 36 663748 0.2 28 56 0.2 36 GalNac₄-11 (3′) 0.6 34 600.1 35 2 44 62 0.1 36 6 38 71 0.1 33

Example 59: Effects of GalNAc₃-1 Conjugated ASOs Targeting FXI In Vivo

The oligonucleotides listed below were tested in a multiple dose studyfor antisense inhibition of FXI in mice. ISIS 404071 was included as anunconjugated standard. Each of the conjugate groups was attached at the3′ terminus of the respective oligonucleotide by a phosphodiester linked2′-deoxyadenosine nucleoside cleavable moiety.

TABLE 49 Modified ASOs targeting FXI SEQ ID ASO Sequence (5′ to 3′)Linkages No. ISIS T_(es)G_(es)G_(es)T_(es)A_(es)A_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds) ^(m)C_(ds) PS 255 404071 T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(es)G_(es)A_(es)G_(es)G_(e) ISIST_(es)G_(es)G_(es)T_(es)A_(es)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)^(m)C_(ds) PS 256 656172 T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(es)G_(es)A_(es)G_(es)G_(eo) A _(do′) -GalNAc ₃ -1 _(a) ISIST_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)^(m)C_(ds) PO/PS 256 656173 T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(eo) A _(do′) -GalNAc ₃ -1 _(a)

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1_(a) was shown previously in Example 9.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) wereinjected subcutaneously twice a week for 3 weeks at the dosage shownbelow with ISIS 404071, 656172, 656173 or with PBS treated control. Eachtreatment group consisted of 4 animals. The mice were sacrificed 72hours following the final administration to determine the liver FXI mRNAlevels using real-time PCR and RIBOGREEN® RNA quantification reagent(Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols.Plasma FXI protein levels were also measured using ELISA. FXI mRNAlevels were determined relative to total RNA (using RIBOGREEN®), priorto normalization to PBS-treated control. The results below are presentedas the average percent of FXI mRNA levels for each treatment group. Thedata was normalized to PBS-treated control and is denoted as “% PBS”.The ED₅₀s were measured using similar methods as described previouslyand are presented below.

TABLE 50 Factor XI mRNA (% Saline) Dose % ASO mg/kg Control ConjugateLinkages Saline 100 none 3 92 none PS ISIS 404071 10 40 30 15 ISIS656172 0.7 74 GalNAc₃-1 PS 2 33 6 9 ISIS 656173 0.7 49 GalNAc₃-1 PO/PS 222 6 1

As illustrated in Table 50, treatment with antisense oligonucleotideslowered FXI mRNA levels in a dose-dependent manner. The oligonucleotidescomprising a 3′-GalNAc₃-1 conjugate group showed substantial improvementin potency compared to the unconjugated antisense oligonucleotide (ISIS404071). Between the two conjugated oligonucleotides an improvement inpotency was further provided by substituting some of the PS linkageswith PO (ISIS 656173).

As illustrated in Table 50a, treatment with antisense oligonucleotideslowered FXI protein levels in a dose-dependent manner. Theoligonucleotides comprising a 3′-GalNAc₃-1 conjugate group showedsubstantial improvement in potency compared to the unconjugatedantisense oligonucleotide (ISIS 404071). Between the two conjugatedoligonucleotides an improvement in potency was further provided bysubstituting some of the PS linkages with PO (ISIS 656173).

TABLE 50a Factor XI protein (% Saline) Dose Protein ASO mg/kg (%Control) Conjugate Linkages Saline 100 none ISIS 404071 3 127 none PS 1032 30 3 ISIS 656172 0.7 70 GalNAc₃-1 PS 2 23 6 1 ISIS 656173 0.7 45GalNAc₃-1 PO/PS 2 6 6 0

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin, total albumin,CRE and BUN were also evaluated. The change in body weights wasevaluated with no significant change from the saline group. ALTs, ASTs,total bilirubin and BUN values are shown in the table below.

TABLE 51 Dosage Total Total ISIS No. mg/kg ALT AST Albumin Bilirubin CREBUN Conjugate Saline 71.8 84.0 3.1 0.2 0.2 22.9 404071 3 152.8 176.0 3.10.3 0.2 23.0 none 10 73.3 121.5 3.0 0.2 0.2 21.4 30 82.5 92.3 3.0 0.20.2 23.0 656172 0.7 62.5 111.5 3.1 0.2 0.2 23.8 GalNac₃-1 (3′) 2 33.051.8 2.9 0.2 0.2 22.0 6 65.0 71.5 3.2 0.2 0.2 23.9 656173 0.7 54.8 90.53.0 0.2 0.2 24.9 GalNac₃-1 (3′) 2 85.8 71.5 3.2 0.2 0.2 21.0 6 114.0101.8 3.3 0.2 0.2 22.7

Example 60: Effects of Conjugated ASOs Targeting SRB-1 In Vitro

The oligonucleotides listed below were tested in a multiple dose studyfor antisense inhibition of SRB-1 in primary mouse hepatocytes. ISIS353382 was included as an unconjugated standard. Each of the conjugategroups were attached at the 3′ or 5′ terminus of the respectiveoligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleosidecleavable moiety.

TABLE 52 Modified ASO targeting SRB-1 SEQ ASO Sequence (5′ to 3′) MotifConjugate ID No. ISIS 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5none 252 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -1 253^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃ -1 _(a) ISIS 655862 G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5GalNAc ₃ -1 253 ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(eo)A _(do′) -GalNAc ₃ -1 _(a) ISIS 661161 GalNAc ₃ -3 _(a-o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds) 5/10/5 GalNAc ₃ -3 254T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 665001 GalNAc ₃ -8 _(a-o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds) 5/10/5 GalNAc₃ -8 254 T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 664078G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) 5/10/5 GalNAc ₃ -9 253^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃ -9 _(a) ISIS 666961 GalNAc ₃ -6 _(a) - _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds) 5/10/5 GalNAc ₃ -6 254T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 664507 GalNAc ₃ -2 _(a) - _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5GalNAc ₃ -2 254 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) ISIS 666881GalNAc ₃ -10 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -10 254^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666224 GalNAc ₃ -5 _(a) - _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -5254 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) ISIS 666981 GalNAc ₃ -7 _(a) - _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) 5/10/5 GalNAc ₃ -7254 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e)

Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-1 a was shown previously in Example 9. Thestructure of GalNAc₃-3a was shown previously in Example 39. Thestructure of GalNAc₃-8a was shown previously in Example 47. Thestructure of GalNAc₃-9a was shown previously in Example 52. Thestructure of GalNAc₃-6a was shown previously in Example 51. Thestructure of GalNAc₃-2a was shown previously in Example 37. Thestructure of GalNAc₃-10a was shown previously in Example 46. Thestructure of GalNAc₃-5a was shown previously in Example 49. Thestructure of GalNAc₃-7a was shown previously in Example 48.

Treatment

The oligonucleotides listed above were tested in vitro in primary mousehepatocyte cells plated at a density of 25,000 cells per well andtreated with 0.03, 0.08, 0.24, 0.74, 2.22, 6.67 or 20 nM modifiedoligonucleotide. After a treatment period of approximately 16 hours, RNAwas isolated from the cells and mRNA levels were measured byquantitative real-time PCR and the SRB-1 mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN®.

The IC₅₀ was calculated using standard methods and the results arepresented in Table 53. The results show that, under free uptakeconditions in which no reagents or electroporation techniques are usedto artificially promote entry of the oligonucleotides into cells, theoligonucleotides comprising a GalNAc conjugate were significantly morepotent in hepatocytes than the parent oligonucleotide (ISIS 353382) thatdoes not comprise a GalNAc conjugate.

TABLE 53 Internucleoside SEQ ID ASO IC₅₀ (nM) linkages Conjugate No.ISIS 353382 190^(a ) PS none 252 ISIS 655861  11^(a) PS GalNAc₃-1  253ISIS 655862 3 PO/PS GalNAc₃-1  253 ISIS 661161 15^(a) PS GalNAc₃-3  254ISIS 665001 20  PS GalNAc₃-8  254 ISIS 664078 55  PS GalNAc₃-9  253 ISIS666961 22^(a) PS GalNAc₃-6  254 ISIS 664507 30  PS GalNAc₃-2  254 ISIS666881 30  PS GalNAc₃-10 254 ISIS 666224 30^(a) PS GalNAc₃-5  254 ISIS666981 40  PS GalNAc₃-7  254 ^(a)Average of multiple nans.

Example 61: Preparation of Oligomeric Compound 175 Comprising GalNAc₃-12

Compound 169 is commercially available. Compound 172 was prepared byaddition of benzyl (perfluorophenyl) glutarate to compound 171. Thebenzyl (perfluorophenyl) glutarate was prepared by adding PFP-TFA andDIEA to 5-(benzyloxy)-5-oxopentanoic acid in DMF. Oligomeric compound175, comprising a GalNAc₃-12 conjugate group, was prepared from compound174 using the general procedures illustrated in Example 46. The GalNAc₃cluster portion of the conjugate group GalNAc₃-12 (GalNAc₃-12_(a)) canbe combined with any cleavable moiety to provide a variety of conjugategroups. In a certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-12(GalNAc₃-12_(a)-CM-) is shown below:

Example 62: Preparation of Oligomeric Compound 180 Comprising GalNAc₃-13

Compound 176 was prepared using the general procedure shown in Example2. Oligomeric compound 180, comprising a GalNAc₃-13 conjugate group, wasprepared from compound 177 using the general procedures illustrated inExample 49. The GalNAc₃ cluster portion of the conjugate groupGalNAc₃-13 (GalNAc₃-13_(a)) can be combined with any cleavable moiety toprovide a variety of conjugate groups. In a certain embodiments, thecleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure ofGalNAc₃-13 (GalNAc₃-13_(a)-CM-) is shown below:

Example 63: Preparation of Oligomeric Compound 188 Comprising GalNAc₃-14

Compounds 181 and 185 are commercially available. Oligomeric compound188, comprising a GalNAc₃-14 conjugate group, was prepared from compound187 using the general procedures illustrated in Example 46. The GalNAc₃cluster portion of the conjugate group GalNAc₃-14 (GalNAc₃-14_(a)) canbe combined with any cleavable moiety to provide a variety of conjugategroups. In certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-14(GalNAc₃-14_(a)-CM-) is shown below:

Example 64: Preparation of Oligomeric Compound 197 Comprising GalNAc₃-15

Compound 189 is commercially available. Compound 195 was prepared usingthe general procedure shown in Example 31. Oligomeric compound 197,comprising a GalNAc₃-15 conjugate group, was prepared from compounds 194and 195 using standard oligonucleotide synthesis procedures. The GalNAc₃cluster portion of the conjugate group GalNAc₃-15 (GalNAc₃-15_(a)) canbe combined with any cleavable moiety to provide a variety of conjugategroups. In certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-15(GalNAc₃-15_(a)-CM-) is shown below:

Example 65: Dose-Dependent Study of Oligonucleotides Comprising a5′-Conjugate Group (Comparison of GalNAc₃-3, 12, 13, 14, and 15)Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 wasincluded as a standard. Each of the GalNAc₃ conjugate groups wasattached at the 5′ terminus of the respective oligonucleotide by aphosphodiester linked 2′-deoxyadenosine nucleoside (cleavable moiety).

TABLE 54 Modified ASOs targeting SRB-1 SEQ ISIS ID No. Sequences (5′ to3′) Conjugate No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) none 252 661161GalNAc ₃ -3 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds) GalNAc₃-3 254 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)671144 GalNAc ₃ -12 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds) GalNAc₃-12 254 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)670061 GalNAc ₃ -13 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds) GalNAc₃-13 254 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)671261 GalNAc ₃ -14 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds) GalNAc₃-14 254 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)671262 GalNAc ₃ -15 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds) GalNAc₃-15 254 T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-3_(a) was shown previously in Example 39. Thestructure of GalNAc₃-12a was shown previously in Example 61. Thestructure of GalNAc₃-13a was shown previously in Example 62. Thestructure of GalNAc₃-14a was shown previously in Example 63. Thestructure of GalNAc₃-15a was shown previously in Example 64.

Treatment

Six to eight week old C57bl6 mice (Jackson Laboratory, Bar Harbor, Me.)were injected subcutaneously once or twice at the dosage shown belowwith ISIS 353382, 661161, 671144, 670061, 671261, 671262, or withsaline. Mice that were dosed twice received the second dose three daysafter the first dose. Each treatment group consisted of 4 animals. Themice were sacrificed 72 hours following the final administration todetermine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN®RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.)according to standard protocols. The results below are presented as theaverage percent of SRB-1 mRNA levels for each treatment group,normalized to the saline control.

As illustrated in Table 55, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. No significantdifferences in target knockdown were observed between animals thatreceived a single dose and animals that received two doses (see ISIS353382 dosages 30 and 2×15 mg/kg; and ISIS 661161 dosages 5 and 2×2.5mg/kg). The antisense oligonucleotides comprising the phosphodiesterlinked GalNAc₃-3, 12, 13, 14, and 15 conjugates showed substantialimprovement in potency compared to the unconjugated antisenseoligonucleotide (ISIS 335382).

TABLE 55 SRB-1 mRNA (% Saline) Dosage SRB-1 mRNA ED₅₀ ISIS No. (mg/kg)(% Saline) (mg/kg) Conjugate Saline n/a 100.0 n/a n/a 353382 3 85.0 22.4none 10 69.5 30 34.2 2 × 15  36.0 661161 0.5 87.4 2.2 GalNAc₃-3  1.559.0 5 25.6 2 × 2.5 27.5 15 17.4 671144 0.5 101.2 3.4 GalNAc₃-12 1.576.1 5 32.0 15 17.6 670061 0.5 94.8 2.1 GalNAc₃-13 1.5 57.8 5 20.7 1513.3 671261 0.5 110.7 4.1 GalNAc₃-14 1.5 81.9 5 39.8 15 14.1 671262 0.5109.4 9.8 GalNAc₃-15 1.5 99.5 5 69.2 15 36.1

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The changes in body weights were evaluated with nosignificant differences from the saline group (data not shown). ALTs,ASTs, total bilirubin and BUN values are shown in Table 56 below.

TABLE 56 Total ISIS Dosage ALT AST Bilirubin BUN No. (mg/kg) (U/L) (U/L)(mg/dL) (mg/dL) Conjugate Saline n/a 28 60 0.1 39 n/a 353382 3 30 77 0.236 none 10 25 78 0.2 36 30 28 62 0.2 35 2 × 15 22 59 0.2 33 661161 0.539 72 0.2 34 GalNAc₃-3 1.5 26 50 0.2 33 5 41 80 0.2 32 2 × 2.5 24 72 0.228 15 32 69 0.2 36 671144 0.5 25 39 0.2 34 GalNAc₃-12 1.5 26 55 0.2 28 548 82 0.2 34 15 23 46 0.2 32 670061 0.5 27 53 0.2 33 GalNAc₃-13 1.5 2445 0.2 35 5 23 58 0.1 34 15 24 72 0.1 31 671261 0.5 69 99 0.1 33GalNAc₃-14 1.5 34 62 0.1 33 5 43 73 0.1 32 15 32 53 0.2 30 671262 0.5 2451 0.2 29 GalNAc₃-15 1.5 32 62 0.1 31 5 30 76 0.2 32 15 31 64 0.1 32

Example 66: Effect of Various Cleavable Moieties on Antisense InhibitionIn Vivo by Oligonucleotides Targeting SRB-1 Comprising a 5′-GalNAc₃Cluster

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Each of the GalNAc₃ conjugategroups was attached at the 5′ terminus of the respective oligonucleotideby a phosphodiester linked nucleoside (cleavable moiety (CM)).

TABLE 57 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 661161 GalNAc ₃ -3 _(a) - _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds) T_(ds) GalNAc₃-3a A_(d) 254 G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 670699 GalNAc ₃-3 _(a) - _(o′) T _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a T_(d) 257G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo)C_(es)T_(es)T_(e) 670700GalNAc ₃ -3 _(a) - _(o′) A _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(e) 254G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e)670701 GalNAc ₃ -3 _(a) - _(o′) T _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a T_(e) 257G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e)671165 GalNAc ₃ -13 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-13a A_(d)254 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e)Capital letters indicate the nucleobase for each nucleoside and ^(m)Cindicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOEmodified nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s”indicates a phosphorothioate internucleoside linkage (PS); “o” indicatesa phosphodiester internucleoside linkage (PO); and “o′” indicates—O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-3_(a) was shown previously in Example 39. Thestructure of GalNAc₃-13a was shown previously in Example 62.

Treatment

Six to eight week old C57bl6 mice (Jackson Laboratory, Bar Harbor, Me.)were injected subcutaneously once at the dosage shown below with ISIS661161, 670699, 670700, 670701, 671165, or with saline. Each treatmentgroup consisted of 4 animals. The mice were sacrificed 72 hoursfollowing the final administration to determine the liver SRB-1 mRNAlevels using real-time PCR and RIBOGREEN® RNA quantification reagent(Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols.The results below are presented as the average percent of SRB-1 mRNAlevels for each treatment group, normalized to the saline control.

As illustrated in Table 58, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. The antisenseoligonucleotides comprising various cleavable moieties all showedsimilar potencies.

TABLE 58 TSRB-1 mRNA (% Saline) Dosage SRB-1 mRNA ISIS No. (mg/kg) (%Saline) GalNAc₃ Cluster CM Saline n/a 100.0 n/a n/a 661161 0.5 87.8GalNAc₃-3a  A_(d) 1.5 61.3 5 33.8 15 14.0 670699 0.5 89.4 GalNAc₃-3a T_(d) 1.5 59.4 5 31.3 15 17.1 670700 0.5 79.0 GalNAc₃-3a  A_(e) 1.5 63.35 32.8 15 17.9 670701 0.5 79.1 GalNAc₃-3a  T_(e) 1.5 59.2 5 35.8 15 17.7671165 0.5 76.4 GalNAc₃-13a A_(d) 1.5 43.2 5 22.6 15 10.0

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The changes in body weights were evaluated with nosignificant differences from the saline group (data not shown). ALTs,ASTs, total bilirubin and BUN values are shown in Table 56 below.

TABLE 59 Total ISIS Dosage ALT AST Bilirubin BUN GalNAc₃ No. (mg/kg)(U/L) (U/L) (mg/dL) (mg/dL) Cluster CM Saline n/a 24 64 0.2 31 n/a n/a661161 0.5 25 64 0.2 31 GalNAc₃-3a A_(d) 1.5 24 50 0.2 32 5 26 55 0.2 2815 27 52 0.2 31 670699 0.5 42 83 0.2 31 GalNAc₃-3a T_(d) 1.5 33 58 0.232 5 26 70 0.2 29 15 25 67 0.2 29 670700 0.5 40 74 0.2 27 GalNAc₃-3aA_(e) 1.5 23 62 0.2 27 5 24 49 0.2 29 15 25 87 0.1 25 670701 0.5 30 770.2 27 GalNAc₃-3a T_(e) 1.5 22 55 0.2 30 5 81 101 0.2 25 15 31 82 0.2 24671165 0.5 44 84 0.2 26 GalNAc₃-13a A_(d) 1.5 47 71 0.1 24 5 33 91 0.226 15 33 56 0.2 29

Example 67: Preparation of Oligomeric Compound 199 Comprising GalNAc₃-16

Oligomeric compound 199, comprising a GalNAc₃-16 conjugate group, isprepared using the general procedures illustrated in Examples 7 and 9.The GalNAc₃ cluster portion of the conjugate group GalNAc₃-16(GalNAc₃-16_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. In certain embodiments, the cleavablemoiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-16(GalNAc₃-16_(a)-CM-) is shown below:

Example 68: Preparation of Oligomeric Compound 200 Comprising GalNAc₃-17

Oligomeric compound 200, comprising a GalNAc₃-17 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-17(GalNAc₃-17_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. In certain embodiments, the cleavablemoiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-17(GalNAc₃-17_(a)-CM-) is shown below:

Example 69: Preparation of Oligomeric Compound 201 Comprising GalNAc₃-18

Oligomeric compound 201, comprising a GalNAc₃-18 conjugate group, wasprepared using the general procedures illustrated in Example 46. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-18(GalNAc₃-18_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. In certain embodiments, the cleavablemoiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-18(GalNAc₃-18_(a)-CM-) is shown below:

Example 70: Preparation of Oligomeric Compound 204 Comprising GalNAc₃-19

Oligomeric compound 204, comprising a GalNAc₃-19 conjugate group, wasprepared from compound 64 using the general procedures illustrated inExample 52. The GalNAc₃ cluster portion of the conjugate groupGalNAc₃-19 (GalNAc₃-19_(a)) can be combined with any cleavable moiety toprovide a variety of conjugate groups. In certain embodiments, thecleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure ofGalNAc₃-19 (GalNAc₃-19_(a)-CM-) is shown below:

Example 71: Preparation of Oligomeric Compound 210 Comprising GalNAc₃-20

Compound 205 was prepared by adding PFP-TFA and DIEA to6-(2,2,2-trifluoroacetamido)hexanoic acid in acetonitrile, which wasprepared by adding triflic anhydride to 6-aminohexanoic acid. Thereaction mixture was heated to 80° C., then lowered to rt. Oligomericcompound 210, comprising a GalNAc₃-20 conjugate group, was prepared fromcompound 208 using the general procedures illustrated in Example 52. TheGalNAc₃ cluster portion of the conjugate group GalNAc₃-20(GalNAc₃-20_(a)) can be combined with any cleavable moiety to provide avariety of conjugate groups. In certain embodiments, the cleavablemoiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-20(GalNAc₃-20_(a)-CM-) is shown below:

Example 72: Preparation of Oligomeric Compound 215 Comprising GalNAc₃-21

Compound 211 is commercially available. Oligomeric compound 215,comprising a GalNAc₃-21 conjugate group, was prepared from compound 213using the general procedures illustrated in Example 52. The GalNAc₃cluster portion of the conjugate group GalNAc₃-21 (GalNAc₃-21_(a)) canbe combined with any cleavable moiety to provide a variety of conjugategroups. In certain embodiments, the cleavable moiety is—P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure of GalNAc₃-21(GalNAc₃-21_(a)-CM-) is shown below:

Example 73: Preparation of Oligomeric Compound 221 Comprising GalNAc₃-22

Compound 220 was prepared from compound 219 using diisopropylammoniumtetrazolide. Oligomeric compound 221, comprising a GalNAc₃-21 conjugategroup, is prepared from compound 220 using the general procedureillustrated in Example 52. The GalNAc₃ cluster portion of the conjugategroup GalNAc₃-22 (GalNAc₃-22_(a)) can be combined with any cleavablemoiety to provide a variety of conjugate groups. In certain embodiments,the cleavable moiety is —P(═O)(OH)-A_(d)-P(═O)(OH)—. The structure ofGalNAc₃-22 (GalNAc₃-22_(a)-CM-) is shown below:

Example 74: Effect of Various Cleavable Moieties on Antisense InhibitionIn Vivo by Oligonucleotides Targeting SRB-1 Comprising a 5′-GalNAc₃Conjugate

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice. Each of the GalNAc₃ conjugategroups was attached at the 5′ terminus of the respectiveoligonucleotide.

TABLE 60 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds) T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) n/a n/a 252 ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)661161 GalNAc ₃ -3 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(d) 254G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)666904 GalNAc ₃ -3 _(a) - _(o′)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a PO 252G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)675441 GalNAc ₃ -17 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-17a A_(d)254 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)675442 GalNAc ₃ -18 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-18a A_(d)254 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)In all tables, capital letters indicate the nucleobase for eachnucleoside and ^(m)C indicates a 5-methyl cytosine. Subscripts: “e”indicates a 2′-MOE modified nucleoside; “d” indicates aβ-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioateinternucleoside linkage (PS); “o” indicates a phosphodiesterinternucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—.Conjugate groups are in bold.

The structure of GalNAc₃-3_(a) was shown previously in Example 39. Thestructure of GalNAc₃-17a was shown previously in Example 68, and thestructure of GalNAc₃-18a was shown in Example 69.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.)were injected subcutaneously once at the dosage shown below with anoligonucleotide listed in Table 60 or with saline. Each treatment groupconsisted of 4 animals. The mice were sacrificed 72 hours following thefinal administration to determine the SRB-1 mRNA levels using real-timePCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc.Eugene, Oreg.) according to standard protocols. The results below arepresented as the average percent of SRB-1 mRNA levels for each treatmentgroup, normalized to the saline control.

As illustrated in Table 61, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner. The antisenseoligonucleotides comprising a GalNAc conjugate showed similar potenciesand were significantly more potent than the parent oligonucleotidelacking a GalNAc conjugate.

TABLE 61 SRB-1 mRNA (% Saline) Dosage SRB-1 mRNA ISIS No. (mg/kg) (%Saline) GalNAc₃ Cluster CM Saline n/a 100.0 n/a n/a 353382 3 79.38 n/an/a 10 68.67 30 40.70 661161 0.5 79.18 GalNAc₃-3a  A_(d) 1.5 75.96 530.53 15 12.52 666904 0.5 91.30 GalNAc₃-3a  PO 1.5 57.88 5 21.22 1516.49 675441 0.5 76.71 GalNAc₃-17a A_(d) 1.5 63.63 5 29.57 15 13.49675442 0.5 95.03 GalNAc₃-18a A_(d) 1.5 60.06 5 31.04 15 19.40

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were measured relative to salineinjected mice using standard protocols. Total bilirubin and BUN werealso evaluated. The change in body weights was evaluated with nosignificant change from the saline group (data not shown). ALTs, ASTs,total bilirubin and BUN values are shown in Table 62 below.

TABLE 62 Total ISIS Dosage ALT AST Bilirubin BUN GalNAc₃ No. (mg/kg)(U/L) (U/L) (mg/dL) (mg/dL) Cluster CM Saline n/a 26 59 0.16 42 n/a n/a353382 3 23 58 0.18 39 n/a n/a 10 28 58 0.16 43 30 20 48 0.12 34 6611610.5 30 47 0.13 35 GalNAc₃-3a A_(d) 1.5 23 53 0.14 37 5 26 48 0.15 39 1532 57 0.15 42 666904 0.5 24 73 0.13 36 GalNAc₃-3a PO 1.5 21 48 0.12 32 519 49 0.14 33 15 20 52 0.15 26 675441 0.5 42 148 0.21 36 GalNAc₃-17aA_(d) 1.5 60 95 0.16 34 5 27 75 0.14 37 15 24 61 0.14 36 675442 0.5 2665 0.15 37 GalNAc₃-18a A_(d) 1.5 25 64 0.15 43 5 27 69 0.15 37 15 30 840.14 37

Example 75: Pharmacokinetic Analysis of Oligonucleotides Comprising a5′-Conjugate Group

The PK of the ASOs in Tables 54, 57 and 60 above was evaluated usingliver samples that were obtained following the treatment proceduresdescribed in Examples 65, 66, and 74. The liver samples were minced andextracted using standard protocols and analyzed by IP-HPLC-MS alongsidean internal standard. The combined tissue level (μg/g) of allmetabolites was measured by integrating the appropriate UV peaks, andthe tissue level of the full-length ASO missing the conjugate (“parent,”which is Isis No. 353382 in this case) was measured using theappropriate extracted ion chromatograms (EIC).

TABLE 63 PK Analysis in Liver Total Tissue Parent ASO ISIS Dosage Levelby Tissue Level GalNAc₃ No. (mg/kg) UV (μg/g) by EIC (μg/g) Cluster CM353382 3 8.9 8.6 n/a n/a 10 22.4 21.0 30 54.2 44.2 661161 5 32.4 20.7GalNAc₃-3a  A_(d) 15 63.2 44.1 671144 5 20.5 19.2 GalNAc₃-12a A_(d) 1548.6 41.5 670061 5 31.6 28.0 GalNAc₃-13a A_(d) 15 67.6 55.5 671261 519.8 16.8 GalNAc₃-14a A_(d) 15 64.7 49.1 671262 5 18.5 7.4 GalNAc₃-15aA_(d) 15 52.3 24.2 670699 5 16.4 10.4 GalNAc₃-3a  T_(d) 15 31.5 22.5670700 5 19.3 10.9 GalNAc₃-3a  A_(e) 15 38.1 20.0 670701 5 21.8 8.8GalNAc₃-3a  T_(e) 15 35.2 16.1 671165 5 27.1 26.5 GalNAc₃-13a A_(d) 1548.3 44.3 666904 5 30.8 24.0 GalNAc₃-3a  PO 15 52.6 37.6 675441 5 25.419.0 GalNAc₃-17a A_(d) 15 54.2 42.1 675442 5 22.2 20.7 GalNAc₃-18a A_(d)15 39.6 29.0

The results in Table 63 above show that there were greater liver tissuelevels of the oligonucleotides comprising a GalNAc₃ conjugate group thanof the parent oligonucleotide that does not comprise a GalNAc₃ conjugategroup (ISIS 353382) 72 hours following oligonucleotide administration,particularly when taking into consideration the differences in dosingbetween the oligonucleotides with and without a GalNAc₃ conjugate group.Furthermore, by 72 hours, 40-98% of each oligonucleotide comprising aGalNAc₃ conjugate group was metabolized to the parent compound,indicating that the GalNAc₃ conjugate groups were cleaved from theoligonucleotides.

Example 76: Preparation of Oligomeric Compound 230 Comprising GalNAc₃-23

Compound 222 is commercially available. 44.48 ml (0.33 mol) of compound222 was treated with tosyl chloride (25.39 g, 0.13 mol) in pyridine (500mL) for 16 hours. The reaction was then evaporated to an oil, dissolvedin EtOAc and washed with water, sat. NaHCO₃, brine, and dried overNa₂SO₄. The ethyl acetate was concentrated to dryness and purified bycolumn chromatography, eluted with EtOAc/hexanes (1:1) followed by 10%methanol in CH₂Cl₂ to give compound 223 as a colorless oil. LCMS and NMRwere consistent with the structure. 10 g (32.86 mmol) of1-Tosyltriethylene glycol (compound 223) was treated with sodium azide(10.68 g, 164.28 mmol) in DMSO (100 mL) at room temperature for 17hours. The reaction mixture was then poured onto water, and extractedwith EtOAc. The organic layer was washed with water three times anddried over Na₂SO₄. The organic layer was concentrated to dryness to give5.3 g of compound 224 (92%). LCMS and NMR were consistent with thestructure. 1-Azidotriethylene glycol (compound 224, 5.53 g, 23.69 mmol)and compound 4 (6 g, 18.22 mmol) were treated with 4 A molecular sieves(5 g), and TMSOTf (1.65 ml, 9.11 mmol) in dichloromethane (100 mL) underan inert atmosphere. After 14 hours, the reaction was filtered to removethe sieves, and the organic layer was washed with sat. NaHCO₃, water,brine, and dried over Na₂SO₄. The organic layer was concentrated todryness and purified by column chromatography, eluted with a gradient of2 to 4% methanol in dichloromethane to give compound 225. LCMS and NMRwere consistent with the structure. Compound 225 (11.9 g, 23.59 mmol)was hydrogenated in EtOAc/Methanol (4:1, 250 mL) over Pearlman'scatalyst. After 8 hours, the catalyst was removed by filtration and thesolvents removed to dryness to give compound 226. LCMS and NMR wereconsistent with the structure.

In order to generate compound 227, a solution ofnitromethanetrispropionic acid (4.17 g, 15.04 mmol) and Hunig's base(10.3 ml, 60.17 mmol) in DMF (100 mL) were treated dropwise withpentaflourotrifluoro acetate (9.05 ml, 52.65 mmol). After 30 minutes,the reaction was poured onto ice water and extracted with EtOAc. Theorganic layer was washed with water, brine, and dried over Na₂SO₄. Theorganic layer was concentrated to dryness and then recrystallized fromheptane to give compound 227 as a white solid. LCMS and NMR wereconsistent with the structure. Compound 227 (1.5 g, 1.93 mmol) andcompound 226 (3.7 g, 7.74 mmol) were stirred at room temperature inacetonitrile (15 mL) for 2 hours. The reaction was then evaporated todryness and purified by column chromatography, eluting with a gradientof 2 to 10% methanol in dichloromethane to give compound 228. LCMS andNMR were consistent with the structure. Compound 228 (1.7 g, 1.02 mmol)was treated with Raney Nickel (about 2 g wet) in ethanol (100 mL) in anatmosphere of hydrogen. After 12 hours, the catalyst was removed byfiltration and the organic layer was evaporated to a solid that was useddirectly in the next step. LCMS and NMR were consistent with thestructure. This solid (0.87 g, 0.53 mmol) was treated withbenzylglutaric acid (0.18 g, 0.8 mmol), HBTU (0.3 g, 0.8 mmol) and DIEA(273.7 μl, 1.6 mmol) in DMF (5 mL). After 16 hours, the DMF was removedunder reduced pressure at 65° C. to an oil, and the oil was dissolved indichloromethane. The organic layer was washed with sat. NaHCO₃, brine,and dried over Na₂SO₄. After evaporation of the organic layer, thecompound was purified by column chromatography and eluted with agradient of 2 to 20% methanol in dichloromethane to give the coupledproduct. LCMS and NMR were consistent with the structure. The benzylester was deprotected with Pearlman's catalyst under a hydrogenatmosphere for 1 hour. The catalyst was them removed by filtration andthe solvents removed to dryness to give the acid. LCMS and NMR wereconsistent with the structure. The acid (486 mg, 0.27 mmol) wasdissolved in dry DMF (3 mL). Pyridine (53.61 μl, 0.66 mmol) was addedand the reaction was purged with argon. Pentaflourotriflouro acetate(46.39 μl, 0.4 mmol) was slowly added to the reaction mixture. The colorof the reaction changed from pale yellow to burgundy, and gave off alight smoke which was blown away with a stream of argon. The reactionwas allowed to stir at room temperature for one hour (completion ofreaction was confirmed by LCMS). The solvent was removed under reducedpressure (rotovap) at 70° C. The residue was diluted with DCM and washedwith 1N NaHSO₄, brine, saturated sodium bicarbonate and brine again. Theorganics were dried over Na₂SO₄, filtered, and were concentrated todryness to give 225 mg of compound 229 as a brittle yellow foam. LCMSand NMR were consistent with the structure.

Oligomeric compound 230, comprising a GalNAc₃-23 conjugate group, wasprepared from compound 229 using the general procedure illustrated inExample 46. The GalNAc₃ cluster portion of the GalNAc₃-23 conjugategroup (GalNAc₃-23_(a)) can be combined with any cleavable moiety toprovide a variety of conjugate groups. The structure of GalNAc₃-23(GalNAc₃-23_(a)-CM) is shown below:

Example 77: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising a GalNAc₃ Conjugate

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of SRB-1 in mice.

TABLE 64 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 661161 GalNAc ₃ -3 _(a) - _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a A_(d) 254 G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 666904 GalNAc ₃-3 _(a) - _(o′)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-3a PO 252G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)673502 GalNAc ₃ -10 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-10a A_(d)254 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e)677844 GalNAc ₃ -9 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-9a A_(d) 254G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)677843 GalNAc ₃ -23 _(a)-_(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) GalNAc₃-23a A_(d)254 G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e)655861 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)GalNAc₃-1a A_(d) 253 ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -1 _(a)677841 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)GalNAc₃-19a A_(d) 253 ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃ -19 _(a)677842 G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds) G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)GalNAc₃-20a A_(d) 253 ^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -20 _(a)

The structure of GalNAc₃-1_(a) was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-9a was shown in Example52, GalNAc₃-10a was shown in Example 46, GalNAc₃-19_(a) was shown inExample 70, GalNAc₃-20_(a) was shown in Example 71, and GalNAc₃-23_(a)was shown in Example 76.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.)were each injected subcutaneously once at a dosage shown below with anoligonucleotide listed in Table 64 or with saline. Each treatment groupconsisted of 4 animals. The mice were sacrificed 72 hours following thefinal administration to determine the SRB-1 mRNA levels using real-timePCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc.Eugene, Oreg.) according to standard protocols. The results below arepresented as the average percent of SRB-1 mRNA levels for each treatmentgroup, normalized to the saline control.

As illustrated in Table 65, treatment with antisense oligonucleotideslowered SRB-1 mRNA levels in a dose-dependent manner.

TABLE 65 SRB-1 mRNA (% Saline) Dosage SRB-1 mRNA ISIS No. (mg/kg) (%Saline) GalNAc₃ Cluster CM Saline n/a 100.0 n/a n/a 0.5 89.18 661161 1.577.02 GalNAc₃-3a  A_(d) 5 29.10 15 12.64 666904 0.5 93.11 GalNAc₃-3a  PO1.5 55.85 5 21.29 15 13.43 673502 0.5 77.75 GalNAc₃-10a A_(d) 1.5 41.055 19.27 15 14.41 677844 0.5 87.65 GalNAc₃-9a  A_(d) 1.5 93.04 5 40.77 1516.95 677843 0.5 102.28 GalNAc₃-23a A_(d) 1.5 70.51 5 30.68 15 13.26655861 0.5 79.72 GalNAc₃-1a  A_(d) 1.5 55.48 5 26.99 15 17.58 677841 0.567.43 GalNAc₃-19a A_(d) 1.5 45.13 5 27.02 15 12.41 677842 0.5 64.13GalNAc₃-20a A_(d) 1.5 53.56 5 20.47 15 10.23

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in serum were also measured using standardprotocols. Total bilirubin and BUN were also evaluated. Changes in bodyweights were evaluated, with no significant change from the saline group(data not shown). ALTs, ASTs, total bilirubin and BUN values are shownin Table 66 below.

TABLE 66 Total ISIS Dosage ALT AST Bilirubin BUN GalNAc₃ No. (mg/kg)(U/L) (U/L) (mg/dL) (mg/dL) Cluster CM Saline n/a 21 45 0.13 34 n/a n/a661161 0.5 28 51 0.14 39 GalNAc₃-3a A_(d) 1.5 23 42 0.13 39 5 22 59 0.1337 15 21 56 0.15 35 666904 0.5 24 56 0.14 37 GalNAc₃-3a PO 1.5 26 680.15 35 5 23 77 0.14 34 15 24 60 0.13 35 673502 0.5 24 59 0.16 34GalNAc₃-10a A_(d) 1.5 20 46 0.17 32 5 24 45 0.12 31 15 24 47 0.13 34677844 0.5 25 61 0.14 37 GalNAc₃-9a A_(d) 1.5 23 64 0.17 33 5 25 58 0.1335 15 22 65 0.14 34 677843 0.5 53 53 0.13 35 GalNAc₃-23a A_(d) 1.5 25 540.13 34 5 21 60 0.15 34 15 22 43 0.12 38 655861 0.5 21 48 0.15 33GalNAc₃-1a A_(d) 1.5 28 54 0.12 35 5 22 60 0.13 36 15 21 55 0.17 30677841 0.5 32 54 0.13 34 GalNAc₃-19a A_(d) 1.5 24 56 0.14 34 5 23 920.18 31 15 24 58 0.15 31 677842 0.5 23 61 0.15 35 GalNAc₃-20a A_(d) 1.524 57 0.14 34 5 41 62 0.15 35 15 24 37 0.14 32

Example 78: Antisense Inhibition In Vivo by Oligonucleotides TargetingAngiotensinogen Comprising a GalNAc₃ Conjugate

The oligonucleotides listed below were tested in a dose-dependent studyfor antisense inhibition of Angiotensinogen (AGT) in normotensiveSprague Dawley rats.

TABLE 67 Modified ASOs targeting AGT SEQ ISIS GalNAc₃ ID No. Sequences(5′ to 3′) Cluster CM No. 552668 ^(m)C_(es)A_(es)^(m)C_(es)T_(es)G_(es)A_(ds)T_(ds) n/a n/a 258T_(ds)T_(ds)T_(ds)T_(ds)G_(ds) ^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(es)G_(es)G_(es)A_(es)T_(e) 669509 ^(m)C_(es)A_(es)^(m)C_(es)T_(es)G_(es)A_(ds)T_(ds) GalNAc₃-1_(a) A_(d) 259T_(ds)T_(ds)T_(ds)T_(ds)G_(ds) ^(m)C_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(es)G_(es)G_(es)A_(es)T_(eo) A _(do′) - GalNAc ₃ -1 _(a)The structure of GalNAc₃-1 a was shown previously in Example 9.

Treatment

Six week old, male Sprague Dawley rats were each injected subcutaneouslyonce per week at a dosage shown below, for a total of three doses, withan oligonucleotide listed in Table 67 or with PBS. Each treatment groupconsisted of 4 animals. The rats were sacrificed 72 hours following thefinal dose. AGT liver mRNA levels were measured using real-time PCR andRIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene,Oreg.) according to standard protocols. AGT plasma protein levels weremeasured using the Total Angiotensinogen ELISA (Catalog # JP27412, IBLInternational, Toronto, ON) with plasma diluted 1:20,000. The resultsbelow are presented as the average percent of AGT mRNA levels in liveror AGT protein levels in plasma for each treatment group, normalized tothe PBS control.

As illustrated in Table 68, treatment with antisense oligonucleotideslowered AGT liver mRNA and plasma protein levels in a dose-dependentmanner, and the oligonucleotide comprising a GalNAc conjugate wassignificantly more potent than the parent oligonucleotide lacking aGalNAc conjugate.

TABLE 68 AGT liver mRNA and plasma protein levels AGT liver AGT plasmaDosage mRNA protein GalNAc₃ ISIS No. (mg/kg) (% PBS) (% PBS) Cluster CMPBS n/a 100 100 n/a n/a 552668 3 95 122 n/a n/a 10 85 97 30 46 79 90 811 669509 0.3 95 70 GalNAc₃-1a A_(d) 1 95 129 3 62 97 10 9 23

Liver transaminase levels, alanine aminotransferase (ALT) and aspartateaminotransferase (AST), in plasma and body weights were also measured attime of sacrifice using standard protocols. The results are shown inTable 69 below.

TABLE 69 Liver transaminase levels and rat body weights Body ISIS DosageALT AST Weight (% GalNAc₃ No. (mg/kg) (U/L) (U/L) of baseline) ClusterCM PBS n/a 51 81 186 n/a n/a 552668 3 54 93 183 n/a n/a 10 51 93 194 3059 99 182 90 56 78 170 669509 0.3 53 90 190 GalNAc₃-1a A_(d) 1 51 93 1923 48 85 189 10 56 95 189

Example 79: Duration of Action In Vivo of Oligonucleotides TargetingAPOC-III Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 70 below were tested in a singledose study for duration of action in mice.

TABLE 70 Modified ASOs targeting APOC-III ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 304801 A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)G_(ds)^(m)C_(ds)T_(es)T_(es) n/a n/a 244 T_(es)A_(es)T_(e) 647535 A_(es)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es) GalNAc₃-1a A_(d) 245T_(es) A _(es) T _(eo) A _(do′) GalNAc ₃ -1 _(a) 663083 GalNAc ₃ -3_(a) - _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-3a A_(d) 260^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) 674449GalNAc ₃-7 _(a) - _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-7a A_(d) 260^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) 674450GalNAc ₃ -10 _(a) - _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-10a A_(d) 260^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e) 674451GalNAc ₃ -13 _(a) - _(o′) A _(do)A_(es)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds) GalNAc₃-13a A_(d) 260^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es)T_(es)A_(es)T_(e)The structure of GalNAc₃-1_(a) was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown inExample 48, GalNAc₃-10_(a) was shown in Example 46, and GalNAc₃-13_(a)was shown in Example 62.

Treatment

Six to eight week old transgenic mice that express human APOC-III wereeach injected subcutaneously once with an oligonucleotide listed inTable 70 or with PBS. Each treatment group consisted of 3 animals. Bloodwas drawn before dosing to determine baseline and at 72 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks following the dose. Plasmatriglyceride and APOC-III protein levels were measured as described inExample 20. The results below are presented as the average percent ofplasma triglyceride and APOC-III levels for each treatment group,normalized to baseline levels, showing that the oligonucleotidescomprising a GalNAc conjugate group exhibited a longer duration ofaction than the parent oligonucleotide without a conjugate group (ISIS304801) even though the dosage of the parent was three times the dosageof the oligonucleotides comprising a GalNAc conjugate group.

TABLE 71 Plasma triglyceride and APOC-III protein levels in transgenicmice Time point Triglyc- APOC-III (days erides protein ISIS Dosage post-(% (% GalNAc₃ No. (mg/kg) dose) baseline) baseline) Cluster CM PBS n/a 397 102 n/a n/a 7 101 98 14 108 98 21 107 107 28 94 91 35 88 90 42 91 105304801 30 3 40 34 n/a n/a 7 41 37 14 50 57 21 50 50 28 57 73 35 68 70 4275 93 647535 10 3 36 37 GalNAc₃-1a A_(d) 7 39 47 14 40 45 21 41 41 28 4262 35 69 69 42 85 102 663083 10 3 24 18 GalNAc₃-3a A_(d) 7 28 23 14 2527 21 28 28 28 37 44 35 55 57 42 60 78 674449 10 3 29 26 GalNAc₃-7aA_(d) 7 32 31 14 38 41 21 44 44 28 53 63 35 69 77 42 78 99 674450 10 333 30 GalNAc₃-10a A_(d) 7 35 34 14 31 34 21 44 44 28 56 61 35 68 70 4283 95 674451 10 3 35 33 GalNAc₃-13a A_(d) 7 24 32 14 40 34 21 48 48 2854 67 35 65 75 42 74 97

Example 80: Antisense Inhibition In Vivo by Oligonucleotides TargetingAlpha-1 Antitrypsin (A1AT) Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 72 below were tested in a study fordose-dependent inhibition of A1AT in mice.

TABLE 72 Modified ASOs targeting A1AT ISIS GalNAc₃ SEQ ID No. Sequences(5′ to 3′) Cluster CM No. 476366 A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(ds)G_(ds)A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es) n/a n/a 261G_(es)G_(es)A_(e) 656326 A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(ds)G_(ds)A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es) GalNAc₃-1aA_(d) 262 G_(es)G_(es)A_(eo)A_(do′)-GalNAc ₃ -1 _(a) 678381 GalNAc ₃ -3_(a) - _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds)A_(ds)GalNAc₃-3a A_(d) 263 A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e)678382 GalNAc ₃ -7 _(a) - _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds)A_(ds)GalNAc₃-7a A_(d) 263 A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e)678383 GalNAc ₃ -10 _(a) - _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds) GalNAc₃-10aA_(d) 263 A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e) 678384GalNAc ₃ -13 _(a) - _(o′) A _(do)A_(es) ^(m)C_(es) ^(m)C_(es)^(m)C_(es)A_(es)A_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(ds)G_(ds) GalNAc₃-13aA_(d) 263 A_(ds)A_(ds)G_(ds)G_(ds)A_(es)A_(es)G_(es)G_(es)A_(e)The structure of GalNAc₃-1_(a) was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown inExample 48, GalNAc₃-10_(a) was shown in Example 46, and GalNAc₃-13_(a)was shown in Example 62.

Treatment

Six week old, male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.)were each injected subcutaneously once per week at a dosage shown below,for a total of three doses, with an oligonucleotide listed in Table 72or with PBS. Each treatment group consisted of 4 animals. The mice weresacrificed 72 hours following the final administration. A1AT liver mRNAlevels were determined using real-time PCR and

RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene,Oreg.) according to standard protocols. A1AT plasma protein levels weredetermined using the Mouse Alpha 1-Antitrypsin ELISA (catalog#41-A1AMS-E01, Alpco, Salem, N.H.). The results below are presented asthe average percent of A1AT liver mRNA and plasma protein levels foreach treatment group, normalized to the PBS control.

As illustrated in Table 73, treatment with antisense oligonucleotideslowered A1AT liver mRNA and A1AT plasma protein levels in adose-dependent manner. The oligonucleotides comprising a GalNAcconjugate were significantly more potent than the parent (ISIS 476366).

TABLE 73 A1AT liver mRNA and plasma protein levels A1AT liver A1ATplasma Dosage mRNA protein GalNAc₃ ISIS No. (mg/kg) (% PBS) (% PBS)Cluster CM PBS n/a 100 100 n/a n/a 476366 5 86 78 n/a n/a 15 73 61 45 3038 656326 0.6 99 90 GalNAc₃-1a  A_(d) 2 61 70 6 15 30 18 6 10 678381 0.6105 90 GalNAc₃-3a  A_(d) 2 53 60 6 16 20 18 7 13 678382 0.6 90 79GalNAc₃-7a  A_(d) 2 49 57 6 21 27 18 8 11 678383 0.6 94 84 GalNAc₃-10aA_(d) 2 44 53 6 13 24 18 6 10 678384 0.6 106 91 GalNAc₃-13a A_(d) 2 6559 6 26 31 18 11 15

Liver transaminase and BUN levels in plasma were measured at time ofsacrifice using standard protocols. Body weights and organ weights werealso measured. The results are shown in Table 74 below. Body weight isshown as % relative to baseline. Organ weights are shown as % of bodyweight relative to the PBS control group.

TABLE 74 ISIS Dosage ALT AST BUN Body weight Liver weight Kidney weightSpleen weight No. (mg/kg) (U/L) (U/L) (mg/dL) (% baseline) (Rel % BW)(Rel % BW) (Rel % BW) PBS n/a 25 51 37 119 100 100 100 476366 5 34 68 35116 91 98 106 15 37 74 30 122 92 101 128 45 30 47 31 118 99 108 123656326 0.6 29 57 40 123 100 103 119 2 36 75 39 114 98 111 106 6 32 67 39125 99 97 122 18 46 77 36 116 102 109 101 678381 0.6 26 57 32 117 93 109110 2 26 52 33 121 96 106 125 6 40 78 32 124 92 106 126 18 31 54 28 11894 103 120 678382 0.6 26 42 35 114 100 103 103 2 25 50 31 117 91 104 1176 30 79 29 117 89 102 107 18 65 112 31 120 89 104 113 678383 0.6 30 6738 121 91 100 123 2 33 53 33 118 98 102 121 6 32 63 32 117 97 105 105 1836 68 31 118 99 103 108 678384 0.6 36 63 31 118 98 103 98 2 32 61 32 11993 102 114 6 34 69 34 122 100 100 96 18 28 54 30 117 98 101 104

Example 81: Duration of Action In Vivo of Oligonucleotides TargetingA1AT Comprising a GalNAc₃ Cluster

The oligonucleotides listed in Table 72 were tested in a single dosestudy for duration of action in mice.

Treatment

Six week old, male C57BL/6 mice were each injected subcutaneously oncewith an oligonucleotide listed in Table 72 or with PBS. Each treatmentgroup consisted of 4 animals. Blood was drawn the day before dosing todetermine baseline and at 5, 12, 19, and 25 days following the dose.Plasma A1AT protein levels were measured via ELISA (see Example 80). Theresults below are presented as the average percent of plasma A1ATprotein levels for each treatment group, normalized to baseline levels.The results show that the oligonucleotides comprising a GalNAc conjugatewere more potent and had longer duration of action than the parentlacking a GalNAc conjugate (ISIS 476366). Furthermore, theoligonucleotides comprising a 5′-GalNAc conjugate (ISIS 678381, 678382,678383, and 678384) were generally even more potent with even longerduration of action than the oligonucleotide comprising a 3′-GalNAcconjugate (ISIS 656326).

TABLE 75 Plasma A1AT protein levels in mice Dosage Time point A1ATGalNAc₃ ISIS No. (mg/kg) (days post-dose) (% baseline) Cluster CM PBSn/a 5 93 n/a n/a 12 93 19 90 25 97 476366 100 5 38 n/a n/a 12 46 19 6225 77 656326 18 5 33 GalNAc₃-1a  A_(d) 12 36 19 51 25 72 678381 18 5 21GalNAc₃-3a  A_(d) 12 21 19 35 25 48 678382 18 5 21 GalNAc₃-7a  A_(d) 1221 19 39 25 60 678383 18 5 24 GalNAc₃-10a A_(d) 12 21 19 45 25 73 67838418 5 29 GalNAc₃-13a A_(d) 12 34 19 57 25 76

Example 82: Antisense Inhibition In Vitro by Oligonucleotides TargetingSRB-1 Comprising a GalNAc₃ Conjugate

Primary mouse liver hepatocytes were seeded in 96 well plates at 15,000cells/well 2 hours prior to treatment. The oligonucleotides listed inTable 76 were added at 2, 10, 50, or 250 nM in Williams E medium andcells were incubated overnight at 37° C. in 5% CO₂. Cells were lysed 16hours following oligonucleotide addition, and total RNA was purifiedusing RNease 3000 BioRobot (Qiagen). SRB-1 mRNA levels were determinedusing real-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. IC₅₀ valueswere determined using Prism 4 software (GraphPad). The results show thatoligonucleotides comprising a variety of different GalNAc conjugategroups and a variety of different cleavable moieties are significantlymore potent in an in vitro free uptake experiment than the parentoligonucleotides lacking a GalNAc conjugate group (ISIS 353382 and666841).

TABLE 76 Inhibition of SRB-1 expression in vitro ISIS GalNAc IC₅₀ SEQNo. Sequence (5′ to 3′) Linkages cluster CM (nM) ID No. 353382 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS n/a n/a 250 252^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 655861 G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS GalNAc₃- A_(d) 40 253^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc₃ -1 _(a) 1_(a) 661161 GalNAc ₃ -3 _(a) - _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 40254 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 3_(a) 661162 GalNAc ₃ -3 _(a) - _(o′) A_(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PSGalNAc₃- A_(d) 8 254 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 3_(a) 664078G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS GalNAc₃- A_(d) 20 253^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃ -9 _(a) 9_(a) 665001 GalNAc ₃ -8 _(a) - _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 70254 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 8_(a) 666224 GalNAc ₃ -5 _(a) - _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PSGalNAc₃- A_(d) 80 254 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 5_(a) 666841G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PO/PS n/a n/a >250 252^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 666881 GalNAc ₃-10 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 30 254^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 10_(a) 666904 GalNAc ₃ -3 _(a) - _(o′)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) PSGalNAc₃- PO 9 252 A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 3_(a) 666924 GalNAc ₃ -3 _(a) - _(o′) T_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PSGalNAc₃- T_(d) 15 257 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 3_(a) 666961GalNAc ₃ -6 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 150 254^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 6_(a) 666981 GalNAc ₃ -7 _(a) - _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PSGalNAc₃- A_(d) 20 254 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 7_(a) 670061GalNAc ₃ -13 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 30 254^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 13_(a) 670699 GalNAc ₃ -3 _(a) - _(o′) T_(do)G_(es) ^(m)C_(es)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PSGalNAc₃- T_(d) 15 257 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 3_(a) 670700GalNAc ₃ -3 _(a) - _(o′) A _(eo)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(e) 30 254^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(eo)^(m)C_(es)T_(es)T 3_(a) 670701 GalNAc ₃ -3 _(a) - _(o′) T _(eo)G_(es)^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- T_(e)25 257 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo)^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 3_(a) 671144 GalNAc ₃ -12 _(a) - _(o′)A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PSGalNAc₃- A_(d) 40 254 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 12_(a) 671165GalNAc ₃ -13 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(d) 8 254^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo)^(m)C_(es)T_(es)T 13_(a) 671261 GalNAc ₃ -14 _(a) - _(o′) A _(do)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃-A_(d) >250 254 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es)^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 14_(a) 671262 GalNAc ₃ -15 _(a) - _(o′)A _(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PSGalNAc₃- A_(d) >250 254 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 15_(a) 673501GalNAc ₃ -7 _(a) - _(o′) A _(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo)^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PS GalNAc₃- A_(d) 30 254^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo)^(m)C_(es)T_(es)T_(e) 7_(a) 673502 GalNAc ₃ -10 _(a) - _(o′) A_(do)G_(es) ^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(eo)A_(ds)G_(ds)T_(ds) PO/PSGalNAc₃- A_(d) 8 254 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(eo) ^(m)C_(eo) ^(m)C_(es)T_(es)T_(e) 10_(a) 675441GalNAc ₃ -17 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) >30 254^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 17_(a) 675442 GalNAc ₃ -18 _(a) - _(o′) A_(do)G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) PSGalNAc₃- A_(d) 20 254 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 18_(a) 677841G_(es) ^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PS GalNAc₃- A_(d) 40 253^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(eo) A _(do′)-GalNAc ₃ -19 _(a) 19_(a) 677842 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) PSGalNAc₃- A_(d) 30 253 ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(eo) A _(do′) -GalNAc ₃ -20 _(a) 20_(a) 677843 GalNAc₃ -23 _(a) - _(o′) A _(do)G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) PS GalNAc₃- A_(d) 40 254^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)^(m)C_(es)T_(es)T_(e) 23_(a)The structure of GalNAc₃-1_(a) was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-5_(a) was shown inExample 49, GalNAc₃-6_(a) was shown in Example 51, GalNAc₃-7_(a) wasshown in Example 48, GalNAc₃-8_(a) was shown in Example 47,GalNAc₃-9_(a) was shown in Example 52, GalNAc₃-10_(a) was shown inExample 46, GalNAc₃-12_(a) was shown in Example 61, GalNAc₃-13_(a) wasshown in Example 62, GalNAc₃-14_(a) was shown in Example 63,GalNAc₃-15_(a) was shown in Example 64, GalNAc₃-17_(a) was shown inExample 68, GalNAc₃-18_(a) was shown in Example 69, GalNAc₃-19_(a) wasshown in Example 70, GalNAc₃-20_(a) was shown in Example 71, andGalNAc₃-23_(a) was shown in Example 76.

Example 83: Antisense Inhibition In Vivo by Oligonucleotides TargetingFactor XI Comprising a GalNAc₃ Cluster

The oligonucleotides listed in Table 77 below were tested in a study fordose-dependent inhibition of Factor XI in mice.

TABLE 77 Modified oligonucleotides targeting Factor XI ISIS GalNAc SEQNo. Sequence (5′ to 3′) cluster CM ID No. 404071T_(es)G_(es)G_(es)T_(es)A_(es)A_(ds)T_(ds) ^(m)C_(ds) ^(m)C_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ds)T_(ds) ^(m)C_(ds)A_(es)G_(es) n/a n/a 255A_(es)G_(es)G_(e) 656173 T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds)^(m)C_(ds) ^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo) GalNAc₃-1_(a) A_(d) 256 A_(es)G_(es)G_(eo) A_(do′) -GalNAc ₃ -1 _(a) 663086 GalNAc ₃ -3 _(a) - _(o′) A_(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-3_(a) A_(d) 264 T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e) 678347 GalNAc ₃ -7 _(a) - _(o′)A _(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds) ^(m)C_(ds)T_(ds) GalNAc₃-7_(a) A_(d) 264 T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e) 678348 GalNAc ₃ -10 _(a) - _(o′)A _(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-10_(a) A_(d) 264 T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e) 678349 GalNAc ₃ -13 _(a) - _(o′)A _(do)T_(es)G_(eo)G_(eo)T_(eo)A_(eo)A_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-13_(a) A_(d) 264 T_(ds)T_(ds)T_(ds)^(m)C_(ds)A_(eo)G_(eo)A_(es)G_(es)G_(e)The structure of GalNAc₃-1_(a) was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown inExample 48, GalNAc₃-10_(a) was shown in Example 46, and GalNAc₃-13_(a)was shown in Example 62.

Treatment

Six to eight week old mice were each injected subcutaneously once perweek at a dosage shown below, for a total of three doses, with anoligonucleotide listed below or with PBS. Each treatment group consistedof 4 animals. The mice were sacrificed 72 hours following the finaldose. Factor XI liver mRNA levels were measured using real-time PCR andnormalized to cyclophilin according to standard protocols. Livertransaminases, BUN, and bilirubin were also measured. The results beloware presented as the average percent for each treatment group,normalized to the PBS control.

As illustrated in Table 78, treatment with antisense oligonucleotideslowered Factor XI liver mRNA in a dose-dependent manner. The resultsshow that the oligonucleotides comprising a GalNAc conjugate were morepotent than the parent lacking a GalNAc conjugate (ISIS 404071).Furthermore, the oligonucleotides comprising a 5′-GalNAc conjugate (ISIS663086, 678347, 678348, and 678349) were even more potent than theoligonucleotide comprising a 3′-GalNAc conjugate (ISIS 656173).

TABLE 78 Factor XI liver mRNA, liver transaminase, BUN, and bilirubinlevels ISIS Dosage Factor XI mRNA ALT AST BUN Bilirubin GalNAc₃ SEQ No.(mg/kg) (% PBS) (U/L) (U/L) (mg/dL) (mg/dL) Cluster ID No. PBS n/a 10063 70 21 0.18 n/a n/a 404071 3 65 41 58 21 0.15 n/a 255 10 33 49 53 230.15 30 17 43 57 22 0.14 656173 0.7 43 90 89 21 0.16 GalNAc₃-1a 256 2 936 58 26 0.17 6 3 50 63 25 0.15 663086 0.7 33 91 169 25 0.16 GalNAc₃-3a264 2 7 38 55 21 0.16 6 1 34 40 23 0.14 678347 0.7 35 28 49 20 0.14GalNAc₃-7a 264 2 10 180 149 21 0.18 6 1 44 76 19 0.15 678348 0.7 39 4354 21 0.16 GalNAc₃-10a 264 2 5 38 55 22 0.17 6 2 25 38 20 0.14 6783490.7 34 39 46 20 0.16 GalNAc₃-13a 264 2 8 43 63 21 0.14 6 2 28 41 20 0.14

Example 84: Duration of Action In Vivo of Oligonucleotides TargetingFactor XI Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 77 were tested in a single dosestudy for duration of action in mice.

Treatment

Six to eight week old mice were each injected subcutaneously once withan oligonucleotide listed in Table 77 or with PBS. Each treatment groupconsisted of 4 animals. Blood was drawn by tail bleeds the day beforedosing to determine baseline and at 3, 10, and 17 days following thedose. Plasma Factor XI protein levels were measured by ELISA usingFactor XI capture and biotinylated detection antibodies from R & DSystems, Minneapolis, Minn. (catalog # AF2460 and # BAF2460,respectively) and the OptEIA Reagent Set B (Catalog #550534, BDBiosciences, San Jose, Calif.). The results below are presented as theaverage percent of plasma Factor XI protein levels for each treatmentgroup, normalized to baseline levels. The results show that theoligonucleotides comprising a GalNAc conjugate were more potent withlonger duration of action than the parent lacking a GalNAc conjugate(ISIS 404071). Furthermore, the oligonucleotides comprising a 5′-GalNAcconjugate (ISIS 663086, 678347, 678348, and 678349) were even morepotent with an even longer duration of action than the oligonucleotidecomprising a 3′-GalNAc conjugate (ISIS 656173).

TABLE 79 Plasma Factor XI protein levels in mice Time point Factor (daysXI ISIS Dosage post- (% GalNAc₃ SEQ No. (mg/kg) dose) baseline) ClusterCM ID No. PBS n/a 3 123 n/a n/a n/a 10 56 17 100 404071 30 3 11 n/a n/a255 10 47 17 52 656173 6 3 1 GalNAc₃-1a A_(d) 256 10 3 17 21 663086 6 31 GalNAc₃-3a A_(d) 264 10 2 17 9 678347 6 3 1 GalNAc₃-7a A_(d) 264 10 117 8 678348 6 3 1 GalNAc₃-10a A_(d) 264 10 1 17 6 678349 6 3 1GalNAc₃-13a A_(d) 264 10 1 17 5

Example 85: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising a GalNAc₃ Conjugate

Oligonucleotides listed in Table 76 were tested in a dose-dependentstudy for antisense inhibition of SRB-1 in mice.

Treatment

Six to eight week old C57BL/6 mice were each injected subcutaneouslyonce per week at a dosage shown below, for a total of three doses, withan oligonucleotide listed in Table 76 or with saline. Each treatmentgroup consisted of 4 animals. The mice were sacrificed 48 hoursfollowing the final administration to determine the SRB-1 mRNA levelsusing real-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) according to standard protocols. The resultsbelow are presented as the average percent of liver SRB-1 mRNA levelsfor each treatment group, normalized to the saline control.

As illustrated in Tables 80 and 81, treatment with antisenseoligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner.

TABLE 80 SRB-1 mRNA in liver Dosage SRB-1 mRNA GalNAc₃ ISIS No. (mg/kg)(% Saline) Cluster CM Saline n/a 100 n/a n/a 655861 0.1 94 GalNAc₃-1a A_(d) 0.3 119 1 68 3 32 661161 0.1 120 GalNAc₃-3a  A_(d) 0.3 107 1 68 326 666881 0.1 107 GalNAc₃-10a A_(d) 0.3 107 1 69 3 27 666981 0.1 120GalNAc₃-7a  A_(d) 0.3 103 1 54 3 21 670061 0.1 118 GalNAc₃-13a A_(d) 0.389 1 52 3 18 677842 0.1 119 GalNAc₃-20a A_(d) 0.3 96 1 65 3 23

TABLE 81 SRB-1 mRNA in liver Dosage SRB-1 mRNA ISIS No. (mg/kg) (%Saline) GalNAc₃ Cluster CM 661161 0.1 107 GalNAc₃-3a  A_(d) 0.3 95 1 533 18 677841 0.1 110 GalNAc₃-19a A_(d) 0.3 88 1 52 3 25

Liver transaminase levels, total bilirubin, BUN, and body weights werealso measured using standard protocols. Average values for eachtreatment group are shown in Table 82 below.

TABLE 82 ISIS Dosage ALT AST Bilirubin BUN Body Weight GalNAc₃ No.(mg/kg) (U/L) (U/L) (mg/dL) (mg/dL) (% baseline) Cluster CM Saline n/a19 39 0.17 26 118 n/a n/a 655861 0.1 25 47 0.17 27 114 GalNAc₃-1a A_(d)0.3 29 56 0.15 27 118 1 20 32 0.14 24 112 3 27 54 0.14 24 115 661161 0.135 83 0.13 24 113 GalNAc₃-3a A_(d) 0.3 42 61 0.15 23 117 1 34 60 0.18 22116 3 29 52 0.13 25 117 666881 0.1 30 51 0.15 23 118 GalNAc₃-10a A_(d)0.3 49 82 0.16 25 119 1 23 45 0.14 24 117 3 20 38 0.15 21 112 666981 0.121 41 0.14 22 113 GalNAc₃-7a A_(d) 0.3 29 49 0.16 24 112 1 19 34 0.15 22111 3 77 78 0.18 25 115 670061 0.1 20 63 0.18 24 111 GalNAc₃-13a A_(d)0.3 20 57 0.15 21 115 1 20 35 0.14 20 115 3 27 42 0.12 20 116 677842 0.120 38 0.17 24 114 GalNAc₃-20a A_(d) 0.3 31 46 0.17 21 117 1 22 34 0.1521 119 3 41 57 0.14 23 118

Example 86: Antisense Inhibition In Vivo by Oligonucleotides TargetingTTR Comprising a GalNAc₃ Cluster

Oligonucleotides listed in Table 83 below were tested in adose-dependent study for antisense inhibition of human transthyretin(TTR) in transgenic mice that express the human TTR gene.

Treatment

Eight week old TTR transgenic mice were each injected subcutaneouslyonce per week for three weeks, for a total of three doses, with anoligonucleotide and dosage listed in the tables below or with PBS. Eachtreatment group consisted of 4 animals. The mice were sacrificed 72hours following the final administration. Tail bleeds were performed atvarious time points throughout the experiment, and plasma TTR protein,ALT, and AST levels were measured and reported in Tables 85-87. Afterthe animals were sacrificed, plasma ALT, AST, and human TTR levels weremeasured, as were body weights, organ weights, and liver human TTR mRNAlevels. TTR protein levels were measured using a clinical analyzer(AU480, Beckman Coulter, CA). Real-time PCR and RIBOGREEN® RNAquantification reagent (Molecular Probes, Inc. Eugene, Oreg.) were usedaccording to standard protocols to determine liver human TTR mRNAlevels. The results presented in Tables 84-87 are the average values foreach treatment group. The mRNA levels are the average values relative tothe average for the PBS group. Plasma protein levels are the averagevalues relative to the average value for the PBS group at baseline. Bodyweights are the average percent weight change from baseline untilsacrifice for each individual treatment group. Organ weights shown arenormalized to the animal's body weight, and the average normalized organweight for each treatment group is then presented relative to theaverage normalized organ weight for the PBS group.

In Tables 84-87, “BL” indicates baseline, measurements that were takenjust prior to the first dose. As illustrated in Tables 84 and 85,treatment with antisense oligonucleotides lowered TTR expression levelsin a dose-dependent manner. The oligonucleotides comprising a GalNAcconjugate were more potent than the parent lacking a GalNAc conjugate(ISIS 420915). Furthermore, the oligonucleotides comprising a GalNAcconjugate and mixed PS/PO internucleoside linkages were even more potentthan the oligonucleotide comprising a GalNAc conjugate and full PSlinkages.

TABLE 83 Oligonucleotides targeting human TTR GalNAc SEQ Isis No.Sequence 5′ to 3′ Linkages cluster CM ID No. 420915 T_(es)^(m)C_(es)T_(es)T_(es)G_(es)G_(ds)T_(ds)T_(ds)A_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds) PS n/a n/a 265 A_(es)T_(es)^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 660261 T_(es)^(m)C_(es)T_(es)T_(es)G_(es)G_(ds)T_(ds)T_(ds)A_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds) PS GalNAc₃-1a A_(d) 266A_(es)T_(es) ^(m)C_(es) ^(m)C_(es) ^(m)C_(eo) A _(do′) -GalNAc ₃ -1 _(a)682883 GalNAc ₃ -3 _(a-o′)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)PS/PO GalNAc₃-3a PO 265 T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 682884 GalNAc ₃ -7 _(a-o′)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)PS/PO GalNAc₃-7a PO 265 T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 682885 GalNAc ₃ -10 _(a-o′)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds) PS/POGalNAc₃-10a PO 265 A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 682886 GalNAc ₃ -13 _(a-o′)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds) PS/POGalNAc₃-13a PO 265 A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 684057 T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds) PS/PO GalNAc₃-19a A_(d) 266A_(eo)T_(eo) ^(m)C_(es) ^(m)C_(es) ^(m)C_(eo) A _(do′) -GalNAc ₃ -19_(a)The legend for Table 85 can be found in Example 74. The structure ofGalNAc₃-1 was shown in Example 9. The structure of GalNAc₃-3_(a) wasshown in Example 39. The structure of GalNAc₃-7_(a) was shown in Example48. The structure of GalNAc₃-10_(a) was shown in Example 46. Thestructure of GalNAc₃-13_(a) was shown in Example 62. The structure ofGalNAc₃-19_(a) was shown in Example 70.

TABLE 84 Antisense inhibition of human TTR in vivo Plasma TTR TTR IsisDosage mRNA protein GalNAc SEQ No. (mg/kg) (% PBS) (% PBS) cluster CM IDNo. PBS n/a 100 100 n/a n/a 420915 6 99 95 n/a n/a 265 20 48 65 60 18 28660261 0.6 113 87 GalNAc₃-1a A_(d) 266 2 40 56 6 20 27 20 9 11

TABLE 85 Antisense inhibition of human TTR in vivo TTR Plasma TTRprotein (% PBS at BL) Isis Dosage mRNA Day 17 GalNAc SEQ No. (mg/kg) (%PBS) BL Day 3 Day 10 (After sac) cluster CM ID No. PBS n/a 100 100 96 90114 n/a n/a 420915 6 74 106 86 76 83 n/a n/a 265 20 43 102 66 61 58 6024 92 43 29 32 682883 0.6 60 88 73 63 68 GalNAc₃-3a PO 265 2 18 75 38 2323 6 10 80 35 11 9 682884 0.6 56 88 78 63 67 GalNAc₃-7a PO 265 2 19 7644 25 23 6 15 82 35 21 24 682885 0.6 60 92 77 68 76 GalNAc₃-10a PO 265 222 93 58 32 32 6 17 85 37 25 20 682886 0.6 57 91 70 64 69 GalNAc₃-13a PO265 2 21 89 50 31 30 6 18 102 41 24 27 684057 0.6 53 80 69 56 62GalNAc₃-19a A_(d) 266 2 21 92 55 34 30 6 11 82 50 18 13

TABLE 86 Transaminase levels, body weight changes, and relative organweights Isis Dosage ALT (U/L) AST (U/L) Body Liver Spleen Kidney SEQ No.(mg/kg) BL Day 3 Day 10 Day 17 BL Day 3 Day 10 Day 17 (% BL) (% PBS) (%PBS) (% PBS) ID No. PBS n/a 33 34 33 24 58 62 67 52 105 100 100 100 n/a420915 6 34 33 27 21 64 59 73 47 115 99 89 91 265 20 34 30 28 19 64 5456 42 111 97 83 89 60 34 35 31 24 61 58 71 58 113 102 98 95 660261 0.633 38 28 26 70 71 63 59 111 96 99 92 266 2 29 32 31 34 61 60 68 61 118100 92 90 6 29 29 28 34 58 59 70 90 114 99 97 95 20 33 32 28 33 64 54 6895 114 101 106 92

TABLE 87 Transaminase levels, body weight changes, and relative organweights Isis Dosage ALT (U/L) AST (U/L) Body Liver Spleen Kidney SEQ No.(mg/kg) BL Day 3 Day 10 Day 17 BL Day 3 Day 10 Day 17 (% BL) (% PBS) (%PBS) (% PBS) ID No. PBS n/a 32 34 37 41 62 78 76 77 104 100 100 100 n/a420915 6 32 30 34 34 61 71 72 66 102 103 102 105 265 20 41 34 37 33 8076 63 54 106 107 135 101 60 36 30 32 34 58 81 57 60 106 105 104 99682883 0.6 32 35 38 40 53 81 74 76 104 101 112 95 265 2 38 39 42 43 7184 70 77 107 98 116 99 6 35 35 41 38 62 79 103 65 105 103 143 97 6828840.6 33 32 35 34 70 74 75 67 101 100 130 99 265 2 31 32 38 38 63 77 66 55104 103 122 100 6 38 32 36 34 65 85 80 62 99 105 129 95 682885 0.6 39 2637 35 63 63 77 59 100 109 109 112 265 2 30 26 38 40 54 56 71 72 102 98111 102 6 27 27 34 35 46 52 56 64 102 98 113 96 682886 0.6 30 40 34 3658 87 54 61 104 99 120 101 265 2 27 26 34 36 51 55 55 69 103 91 105 92 640 28 34 37 107 54 61 69 109 100 102 99 684057 0.6 35 26 33 39 56 51 5169 104 99 110 102 266 2 33 32 31 40 54 57 56 87 103 100 112 97 6 39 3335 40 67 52 55 92 98 104 121 108

Example 87: Duration of Action In Vivo by Single Doses ofOligonucleotides Targeting TTR Comprising a GalNAc₃ Cluster

ISIS numbers 420915 and 660261 (see Table 83) were tested in a singledose study for duration of action in mice. ISIS numbers 420915, 682883,and 682885 (see Table 83) were also tested in a single dose study forduration of action in mice.

Treatment

Eight week old, male transgenic mice that express human TTR were eachinjected subcutaneously once with 100 mg/kg ISIS No. 420915 or 13.5mg/kg ISIS No. 660261. Each treatment group consisted of 4 animals. Tailbleeds were performed before dosing to determine baseline and at days 3,7, 10, 17, 24, and 39 following the dose. Plasma TTR protein levels weremeasured as described in Example 86. The results below are presented asthe average percent of plasma TTR levels for each treatment group,normalized to baseline levels.

TABLE 88 Plasma TTR protein levels Time point (days TTR ISIS Dosagepost- (% GalNAc₃ SEQ No. (mg/kg) dose) baseline) Cluster CM ID No.420915 100 3 30 n/a n/a 265 7 23 10 35 17 53 24 75 39 100 660261 13.5 327 GalNAc₃-1a A_(d) 266 7 21 10 22 17 36 24 48 39 69

Treatment

Female transgenic mice that express human TTR were each injectedsubcutaneously once with 100 mg/kg ISIS No. 420915, 10.0 mg/kg ISIS No.682883, or 10.0 mg/kg 682885. Each treatment group consisted of 4animals. Tail bleeds were performed before dosing to determine baselineand at days 3, 7, 10, 17, 24, and 39 following the dose. Plasma TTRprotein levels were measured as described in Example 86. The resultsbelow are presented as the average percent of plasma TTR levels for eachtreatment group, normalized to baseline levels.

TABLE 89 Plasma TTR protein levels Time point (days TTR ISIS Dosagepost- (% GalNAc₃ SEQ No. (mg/kg) dose) baseline) Cluster CM ID No.420915 100 3 48 n/a n/a 265 7 48 10 48 17 66 31 80 682883 10.0 3 45GalNAc₃-3a PO 265 7 37 10 38 17 42 31 65 682885 10.0 3 40 GalNAc₃-10a PO265 7 33 10 34 17 40 31 64The results in Tables 88 and 89 show that the oligonucleotidescomprising a GalNAc conjugate are more potent with a longer duration ofaction than the parent oligonucleotide lacking a conjugate (ISIS420915).

Example 88: Splicing Modulation In Vivo by Oligonucleotides TargetingSMN Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 90 were tested for splicingmodulation of human survival of motor neuron (SMN) in mice.

TABLE 90 Modified ASOs targeting SMN ISIS GalNAc₃ SEQ No. Sequences (5′to 3′) Cluster CM ID No. 387954 A_(es)T_(es)T_(es) ^(m)C_(es)A_(es)^(m)C_(es)T_(es)T_(es)T_(es)^(m)C_(es)A_(es)T_(es)A_(es)A_(es)T_(es)G_(es)^(m)C_(es)T_(es)G_(es)G_(e) n/a n/a 267 699819GalNAc₃-7_(a-o’)A_(es)T_(es)T_(es) ^(m)C_(es)A_(es)^(m)C_(es)T_(es)T_(es)T_(es) ^(m)C_(es)A_(es)T_(es)A_(es)A_(es)GalNAc₃-7a PO 267 T_(es)G_(es) ^(m)C_(es)T_(es)G_(es)G_(e) 699821

GalNAc₃-7a PO 267

700000

GalNAc₃-1a Ad 268

703421 X-ATT^(m)CA^(m)CTTT^(m)CATAATG^(m)CTGG n/a n/a 267 703422GalNAc₃-7_(b)-X-ATT^(m)CA^(m)CTTT^(m)CATAATG^(m)CTGG GalNAc₃-7b n/a 267The structure of GalNAc₃-7_(a) was shown previously in Example 48. “X”indicates a 5′ primary amine generated by Gene Tools (Philomath, Oreg.),and GalNAc₃-7_(b) indicates the structure of GalNAc₃-7_(a) lacking the—NH—C₆—O portion of the linker as shown below:

ISIS numbers 703421 and 703422 are morpholino oligonucleotides, whereineach nucleotide of the two oligonucleotides is a morpholino nucleotide.

Treatment

Six week old transgenic mice that express human SMN were injectedsubcutaneously once with an oligonucleotide listed in Table 91 or withsaline. Each treatment group consisted of 2 males and 2 females. Themice were sacrificed 3 days following the dose to determine the liverhuman SMN mRNA levels both with and without exon 7 using real-time PCRaccording to standard protocols. Total RNA was measured using Ribogreenreagent. The SMN mRNA levels were normalized to total mRNA, and furthernormalized to the averages for the saline treatment group. The resultingaverage ratios of SMN mRNA including exon 7 to SMN mRNA missing exon 7are shown in Table 91. The results show that fully modifiedoligonucleotides that modulate splicing and comprise a GalNAc conjugateare significantly more potent in altering splicing in the liver than theparent oligonucleotides lacking a GlaNAc conjugate. Furthermore, thistrend is maintained for multiple modification chemistries, including2′-MOE and morpholino modified oligonucleotides.

TABLE 91 Effect of oligonucleotides targeting human SMN in vivo ISISDose GalNAc₃ SEQ No. (mg/kg) +Exon 7/−Exon 7 Cluster CM ID No. Salinen/a 1.00 n/a n/a n/a 387954 32 1.65 n/a n/a 267 387954 288 5.00 n/a n/a267 699819 32 7.84 GalNAc₃-7a PO 267 699821 32 7.22 GalNAc₃-7a PO 267700000 32 6.91 GalNAc₃-1a A_(d) 268 703421 32 1.27 n/a n/a 267 703422 324.12 GalNAc₃-7b n/a 267

Example 89: Antisense Inhibition In Vivo by Oligonucleotides TargetingApolipoprotein A (Apo(a)) Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 92 below were tested in a study fordose-dependent inhibition of Apo(a) in transgenic mice.

TABLE 92 Modified ASOs targeting Apo(a) ISIS GalNAc₃ SEQ ID No.Sequences (5′ to 3′) Cluster CM No. 494372 T_(es)G_(es) ^(m)C_(es)T_(es)^(m)C_(es) ^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)^(m)C_(ds) n/a n/a 277 T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681257GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 277 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e)The structure of GalNAc₃-7_(a) was shown in Example 48.

Treatment

Eight week old, female C57BL/6 mice (Jackson Laboratory, Bar Harbor,Me.) were each injected subcutaneously once per week at a dosage shownbelow, for a total of six doses, with an oligonucleotide listed in Table92 or with PBS. Each treatment group consisted of 3-4 animals. Tailbleeds were performed the day before the first dose and weekly followingeach dose to determine plasma Apo(a) protein levels. The mice weresacrificed two days following the final administration. Apo(a) livermRNA levels were determined using real-time PCR and RIBOGREEN® RNAquantification reagent (Molecular Probes, Inc. Eugene, Oreg.) accordingto standard protocols. Apo(a) plasma protein levels were determinedusing ELISA, and liver transaminase levels were determined. The mRNA andplasma protein results in Table 93 are presented as the treatment groupaverage percent relative to the PBS treated group. Plasma protein levelswere further normalized to the baseline (BL) value for the PBS group.Average absolute transaminase levels and body weights (% relative tobaseline averages) are reported in Table 94.

As illustrated in Table 93, treatment with the oligonucleotides loweredApo(a) liver mRNA and plasma protein levels in a dose-dependent manner.Furthermore, the oligonucleotide comprising the GalNAc conjugate wassignificantly more potent with a longer duration of action than theparent oligonucleotide lacking a GalNAc conjugate. As illustrated inTable 94, transaminase levels and body weights were unaffected by theoligonucleotides, indicating that the oligonucleotides were welltolerated.

TABLE 93 Apo(a) liver mRNA and plasma protein levels ISIS Dosage Apo(a)mRNA Apo(a) plasma protein (% PBS) No. (mg/kg) (% PBS) BL Week 1 Week 2Week 3 Week 4 Week 5 Week 6 PBS n/a 100 100 120 119 113 88 121 97 4943723 80 84 89 91 98 87 87 79 10 30 87 72 76 71 57 59 46 30 5 92 54 28 10 79 7 681257 0.3 75 79 76 89 98 71 94 78 1 19 79 88 66 60 54 32 24 3 2 8252 17 7 4 6 5 10 2 79 17 6 3 2 4 5

TABLE 94 Dosage ALT AST Body weight ISIS No. (mg/kg) (U/L) (U/L) (%baseline) PBS n/a 37 54 103 494372 3 28 68 106 10 22 55 102 30 19 48 103681257 0.3 30 80 104 1 26 47 105 3 29 62 102 10 21 52 107

Example 90: Antisense Inhibition In Vivo by Oligonucleotides TargetingTTR Comprising a GalNAc₃ Cluster

Oligonucleotides listed in Table 95 below were tested in adose-dependent study for antisense inhibition of human transthyretin(TTR) in transgenic mice that express the human TTR gene.

Treatment

TTR transgenic mice were each injected subcutaneously once per week forthree weeks, for a total of three doses, with an oligonucleotide anddosage listed in Table 96 or with PBS. Each treatment group consisted of4 animals. Prior to the first dose, a tail bleed was performed todetermine plasma TTR protein levels at baseline (BL). The mice weresacrificed 72 hours following the final administration. TTR proteinlevels were measured using a clinical analyzer (AU480, Beckman Coulter,CA). Real-time PCR and RIBOGREEN® RNA quantification reagent (MolecularProbes, Inc. Eugene, Oreg.) were used according to standard protocols todetermine liver human TTR mRNA levels. The results presented in Table 96are the average values for each treatment group. The mRNA levels are theaverage values relative to the average for the PBS group. Plasma proteinlevels are the average values relative to the average value for the PBSgroup at baseline. “BL” indicates baseline, measurements that were takenjust prior to the first dose. As illustrated in Table 96, treatment withantisense oligonucleotides lowered TTR expression levels in adose-dependent manner. The oligonucleotides comprising a GalNAcconjugate were more potent than the parent lacking a GalNAc conjugate(ISIS 420915), and oligonucleotides comprising a phosphodiester ordeoxyadenosine cleavable moiety showed significant improvements inpotency compared to the parent lacking a conjugate (see ISIS numbers682883 and 666943 vs 420915 and see Examples 86 and 87).

TABLE 95 Oligonucleotides targeting human TTR GalNAc SEQ Isis No.Sequence 5′ to 3′ Linkages cluster CM ID No. 420915 T_(es)^(m)C_(es)T_(es)T_(es)G_(es)G_(ds)T_(ds)T_(ds)A_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds) PS n/a n/a 265 A_(es)T_(es)^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682883 GalNAc ₃ -3 _(a-o′)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) ^(m)C_(ds)A_(ds)PS/PO GalNAc₃-3a PO 265 T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 666943 GalNAc ₃ -3 _(a-o′)A_(do)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/PO GalNAc₃-3aA_(d) 269 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo)^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682887 GalNAc₃ -7 _(a-o′) A _(do)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/PO GalNAc₃-7aA_(d) 269 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo)^(m)C_(es) ^(m)C_(es) ^(m)C_(e) 682888 GalNAc ₃ -10 _(a-o′) A_(do)T_(es) ^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/POGalNAc₃-10a A_(d) 269^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo) ^(m)C_(es)^(m)C_(es) ^(m)C_(e) 682889 GalNAc ₃ -13 _(a-o′) A _(do)T_(es)^(m)C_(eo)T_(eo)T_(eo)G_(eo)G_(ds)T_(ds)T_(ds)A_(ds) PS/PO GalNAc₃-13aA_(d) 269 ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)A_(ds)A_(eo)T_(eo)^(m)C_(es) ^(m)C_(es) ^(m)C_(e)The legend for Table 95 can be found in Example 74. The structure ofGalNAc₃-3_(a) was shown in Example 39. The structure of GalNAc₃-7_(a)was shown in Example 48. The structure of GalNAc₃-10_(a) was shown inExample 46. The structure of GalNAc₃-13_(a) was shown in Example 62.

TABLE 96 Antisense inhibition of human TTR in vivo Dosage TTR mRNA TTRprotein Isis No. (mg/kg) (% PBS) (% BL) GalNAc cluster CM PBS n/a 100124 n/a n/a 420915 6 69 114 n/a n/a 20 71 86 60 21 36 682883 0.6 61 73GalNAc₃-3a  PO 2 23 36 6 18 23 666943 0.6 74 93 GalNAc₃-3a  A_(d) 2 3357 6 17 22 682887 0.6 60 97 GalNAc₃-7a  A_(d) 2 36 49 6 12 19 682888 0.665 92 GalNAc₃-10a A_(d) 2 32 46 6 17 22 682889 0.6 72 74 GalNAc₃-13aA_(d) 2 38 45 6 16 18

Example 91: Antisense Inhibition In Vivo by Oligonucleotides TargetingFactor VII Comprising a GalNAc₃ Conjugate in Non-Human Primates

Oligonucleotides listed in Table 97 below were tested in a non-terminal,dose escalation study for antisense inhibition of Factor VII in monkeys.

Treatment

Non-naïve monkeys were each injected subcutaneously on days 0, 15, and29 with escalating doses of an oligonucleotide listed in Table 97 orwith PBS. Each treatment group consisted of 4 males and 1 female. Priorto the first dose and at various time points thereafter, blood drawswere performed to determine plasma Factor VII protein levels. Factor VIIprotein levels were measured by ELISA. The results presented in Table 98are the average values for each treatment group relative to the averagevalue for the PBS group at baseline (BL), the measurements taken justprior to the first dose. As illustrated in Table 98, treatment withantisense oligonucleotides lowered Factor VII expression levels in adose-dependent manner, and the oligonucleotide comprising the GalNAcconjugate was significantly more potent in monkeys compared to theoligonucleotide lacking a GalNAc conjugate.

TABLE 97 Oligonucleotides targeting Factor VII GalNAc SEQ Isis No.Sequence 5′ to 3′ Linkages cluster CM ID No. 407935 A_(es)T_(es)G_(es)^(m)C_(es)A_(es)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)A_(ds)T_(ds)G_(ds)^(m)C_(ds)T_(ds) PS n/a n/a 270 T_(es) ^(m)C_(es)T_(es)G_(es)A_(e)686892 GalNAc ₃ -10 _(a-o′)A_(es)T_(es)G_(es)^(m)C_(es)A_(es)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) PS GalNAc₃-10a PO 270A_(ds)T_(ds)G_(ds) ^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es)T_(es)G_(es)A_(e)The legend for Table 97 can be found in Example 74. The structure ofGalNAc₃-10_(a) was shown in Example 46.

TABLE 98 Factor VII plasma protein levels ISIS No. Day Dose (mg/kg)Factor VII (% BL) 407935 0 n/a 100 15 10 87 22 n/a 92 29 30 77 36 n/a 4643 n/a 43 686892 0 3 100 15 10 56 22 n/a 29 29 30 19 36 n/a 15 43 n/a 11

Example 92: Antisense Inhibition in Primary Hepatocytes by AntisenseOligonucleotides Targeting Apo-CIII Comprising a GalNAc₃ Conjugate

Primary mouse hepatocytes were seeded in 96-well plates at 15,000 cellsper well, and the oligonucleotides listed in Table 99, targeting mouseApoC-III, were added at 0.46, 1.37, 4.12, or 12.35, 37.04, 111.11, or333.33 nM or 1.00 μM. After incubation with the oligonucleotides for 24hours, the cells were lysed and total RNA was purified using RNeasy(Qiagen). ApoC-III mRNA levels were determined using real-time PCR andRIBOGREEN® RNA quantification reagent (Molecular Probes, Inc.) accordingto standard protocols. IC₅₀ values were determined using Prism 4software (GraphPad). The results show that regardless of whether thecleavable moiety was a phosphodiester or a phosphodiester-linkeddeoxyadensoine, the oligonucleotides comprising a GalNAc conjugate weresignificantly more potent than the parent oligonucleotide lacking aconjugate.

TABLE 99 Inhibition of mouse APOC-III expression in mouse primaryhepatocytes ISIS IC₅₀ SEQ No. Sequence (5′ to 3′) CM (nM) ID No. 440670^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es)A_(es)G_(es) ^(m)C_(es)A_(e) n/a 13.20 271 661180^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) A_(d) 1.40 272 A_(es)G_(es) ^(m)C_(es)A_(eo) A _(do′) -GalNAc₃ -1 _(a) 680771 GalNAc ₃ -3 _(a-o′) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) PO 0.70 271 A_(es)G_(es) ^(m)C_(es)A_(e) 680772 GalNAc ₃ -7_(a-o′) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) PO 1.70 271 A_(es)G_(es) ^(m)C_(es)A_(e) 680773 GalNAc ₃ -10_(a-o′) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) PO 2.00 271 A_(es)G_(es) ^(m)C_(es)A_(e) 680774 GalNAc ₃ -13_(a-o′) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) PO 1.50 271 A_(es)G_(es) ^(m)C_(es)A_(e) 681272 GalNAc ₃ -3_(a-o′) ^(m)C_(es)A_(eo)G_(eo)^(m)C_(eo)T_(eo)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(eo) PO <0.46 271 A_(eo)G_(es) ^(m)C_(es)A_(e) 681273 GalNAc ₃ -3_(a)-_(o′) A _(do) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)A_(d) 1.10 273 ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)A_(e) 683733^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds)^(m)C_(es) A_(d) 2.50 272 A_(es)G_(es) ^(m)C_(es)A_(eo) A _(do′) -GalNAc₃ -19 _(a)The structure of GalNAc₃-1_(a) was shown previously in Example 9,GalNAc₃-3_(a) was shown in Example 39, GalNAc₃-7_(a) was shown inExample 48, GalNAc₃-10_(a) was shown in Example 46, GalNAc₃-13_(a) wasshown in Example 62, and GalNAc₃-19_(a) was shown in Example 70.

Example 93: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising Mixed Wings and a 5′-GalNAc₃ Conjugate

The oligonucleotides listed in Table 100 were tested in a dose-dependentstudy for antisense inhibition of SRB-1 in mice.

TABLE 100 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 449093 T_(ks)T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) A_(ds)T_(ds) G_(ds) A_(ds)^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) n/a n/a 274 699806 GalNAc ₃-3 _(a) - _(o′)T_(ks)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds) G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-3a PO 274 T_(ds)T_(ks)^(m)C_(ks) ^(m)C_(k) 699807 GalNAc ₃ -7 _(a) - _(o′)T_(ks)T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) A_(ds)T_(ds) G_(ds)A_(ds)^(m)C_(ds) GalNAc₃-7a PO 274 T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(k) 699809GalNAc ₃ -7 _(a) - _(o′) T_(ks)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds) A_(ds)T_(ds) G_(ds) A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 274T_(ds)T_(es) ^(m)C_(es) ^(m)C_(e) 699811 GalNAc ₃ -7 _(a) -_(o′)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 274 T_(ds)T_(ks) ^(m)C_(ks)^(m)C_(k) 699813 GalNAc ₃ -7 _(a) - _(o′)T_(ks)T_(ds)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds) A_(ds)T_(ds) G_(ds)A_(ds)^(m)C_(ds) GalNAc₃-7a PO 274 T_(ds)T_(ks) ^(m)C_(ds) ^(m)C_(k) 699815GalNAc ₃ -7 _(a) - _(o′)T_(es)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds) A_(ds)T_(ds) G_(ds)A_(ds) ^(m)C_(ds) GalNAc₃-7a PO 274T_(ds)T_(ks) ^(m)C_(ks) ^(m)C_(e)The structure of GalNAc₃-3_(a) was shown previously in Example 39, andthe structure of GalNAc₃-7a was shown previously in Example 48.Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicatesβ-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃ bicyclic nucleoside(cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “o”indicates phosphodiester internucleoside linkages (PO). Superscript “m”indicates 5-methylcytosines.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.)were injected subcutaneously once at the dosage shown below with anoligonucleotide listed in Table 100 or with saline. Each treatment groupconsisted of 4 animals. The mice were sacrificed 72 hours following thefinal administration. Liver SRB-1 mRNA levels were measured usingreal-time PCR. SRB-1 mRNA levels were normalized to cyclophilin mRNAlevels according to standard protocols. The results are presented as theaverage percent of SRB-1 mRNA levels for each treatment group relativeto the saline control group. As illustrated in Table 101, treatment withantisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependentmanner, and the gapmer oligonucleotides comprising a GalNAc conjugateand having wings that were either full cEt or mixed sugar modificationswere significantly more potent than the parent oligonucleotide lacking aconjugate and comprising full cEt modified wings.

Body weights, liver transaminases, total bilirubin, and BUN were alsomeasured, and the average values for each treatment group are shown inTable 101. Body weight is shown as the average percent body weightrelative to the baseline body weight (% BL) measured just prior to theoligonucleotide dose.

TABLE 101 SRB-1 mRNA, ALT, AST, BUN, and total bilirubin levels and bodyweights SRB-1 ISIS Dosage mRNA ALT AST Body weight No. (mg/kg) (% PBS)(U/L) (U/L) Bil BUN (% BL) PBS n/a 100 31 84 0.15 28 102 449093 1 111 1848 0.17 31 104 3 94 20 43 0.15 26 103 10 36 19 50 0.12 29 104 699806 0.1114 23 58 0.13 26 107 0.3 59 21 45 0.12 27 108 1 25 30 61 0.12 30 104699807 0.1 121 19 41 0.14 25 100 0.3 73 23 56 0.13 26 105 1 24 22 690.14 25 102 699809 0.1 125 23 57 0.14 26 104 0.3 70 20 49 0.10 25 105 133 34 62 0.17 25 107 699811 0.1 123 48 77 0.14 24 106 0.3 94 20 45 0.1325 101 1 66 57 104 0.14 24 107 699813 0.1 95 20 58 0.13 28 104 0.3 98 2261 0.17 28 105 1 49 19 47 0.11 27 106 699815 0.1 93 30 79 0.17 25 1050.3 64 30 61 0.12 26 105 1 24 18 41 0.14 25 106

Example 94: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising 2′-Sugar Modifications and a 5′-GalNAc₃ Conjugate

The oligonucleotides listed in Table 102 were tested in a dose-dependentstudy for antisense inhibition of SRB-1 in mice.

TABLE 102 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No. 353382 G_(es) ^(m)C_(es)T_(es)T_(es)^(m)C_(es)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es) n/a n/a 252 T_(es)T_(e)700989 G_(ms)C_(ms)U_(ms)U_(ms)C_(ms)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)U_(ms)C_(ms)C_(ms)n/a n/a 275 U_(ms)U_(m) 666904 GalNAc ₃ -3 _(a) - _(o′)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(es)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₃-3a PO 252^(m)C_(ds)T_(ds)T_(es) ^(m)C_(es) ^(m)C_(es)T_(es)T_(e) 700991 GalNAc ₃-7 _(a)-_(o′)G_(ms)C_(ms)U_(ms)U_(ms)C_(ms)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds) GalNAc₃-7a PO 275 A_(ds)^(m)C_(ds)T_(ds)U_(ms)C_(ms)C_(ms)U_(ms)U_(m)Subscript “m” indicates a 2′-O-methyl modified nucleoside. See Example74 for complete table legend. The structure of GalNAc₃-3_(a) was shownpreviously in Example 39, and the structure of GalNAc₃-7a was shownpreviously in Example 48.

Treatment

The study was completed using the protocol described in Example 93.Results are shown in Table 103 below and show that both the 2′-MOE and2′-OMe modified oligonucleotides comprising a GalNAc conjugate weresignificantly more potent than the respective parent oligonucleotideslacking a conjugate. The results of the body weights, livertransaminases, total bilirubin, and BUN measurements indicated that thecompounds were all well tolerated.

TABLE 103 SRB-1 mRNA ISIS No. Dosage (mg/kg) SRB-1 mRNA (% PBS) PBS n/a100 353382 5 116 15 58 45 27 700989 5 120 15 92 45 46 666904 1 98 3 4510 17 700991 1 118 3 63 10 14

Example 95: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising Bicyclic Nucleosides and a 5′-GalNAc₃ Conjugate

The oligonucleotides listed in Table 104 were tested in a dose-dependentstudy for antisense inhibition of SRB-1 in mice.

TABLE 104 Modified ASOs targeting SRB-1 ISIS GalNAc₃ SEQ No. Sequences(5′ to 3′) Cluster CM ID No 440762 T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) n/an/a 246 666905 GalNAc ₃ -3 _(a) - _(o′)T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds) A_(ds)^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) GalNAc₃-3_(a) PO 246 699782 GalNAc ₃ -7_(a) - _(o′)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k)GalNAc₃-7_(a) PO 246 699783 GalNAc ₃ -3 _(a) - _(o′)T_(ls)^(m)C_(ls)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ls) ^(m)C_(l) GalNAc₃-3_(a) PO 246 653621 T_(ls)^(m)C_(ls)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ls) ^(m)C_(lo) A _(do′) -GalNAc ₃ -1 _(a)GalNAc₃-1_(a) A_(d) 247 439879 T_(gs) ^(m)C_(gs)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(d) G_(ds)A_(ds) ^(m)C_(ds)T_(ds)T_(gs) ^(m)C_(g) n/an/a 246 699789 GalNAc ₃ -3 _(a) - _(o′)T_(gs)^(m)C_(gs)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(d) G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(gs) ^(m)C_(g) GalNAc₃-3_(a) PO 246Subscript “g” indicates a fluoro-HNA nucleoside, subscript “1” indicatesa locked nucleoside comprising a 2′-O—CH₂-4′ bridge. See the Example 74table legend for other abbreviations. The structure of GalNAc₃-1_(a) wasshown previously in Example 9, the structure of GalNAc₃-3_(a) was shownpreviously in Example 39, and the structure of GalNAc₃-7a was shownpreviously in Example 48.

Treatment

The study was completed using the protocol described in Example 93.Results are shown in Table 105 below and show that oligonucleotidescomprising a GalNAc conjugate and various bicyclic nucleosidemodifications were significantly more potent than the parentoligonucleotide lacking a conjugate and comprising bicyclic nucleosidemodifications. Furthermore, the oligonucleotide comprising a GalNAcconjugate and fluoro-HNA modifications was significantly more potentthan the parent lacking a conjugate and comprising fluoro-HNAmodifications. The results of the body weights, liver transaminases,total bilirubin, and BUN measurements indicated that the compounds wereall well tolerated.

TABLE 105 SRB-1 mRNA, ALT, AST, BUN, and total bilirubin levels and bodyweights ISIS No. Dosage (mg/kg) SRB-1 mRNA (% PBS) PBS n/a 100 440762 1104 3 65 10 35 666905 0.1 105 0.3 56 1 18 699782 0.1 93 0.3 63 1 15699783 0.1 105 0.3 53 1 12 653621 0.1 109 0.3 82 1 27 439879 1 96 3 7710 37 699789 0.1 82 0.3 69 1 26

Example 96: Plasma Protein Binding of Antisense OligonucleotidesComprising a GalNAc₃ Conjugate Group

Oligonucleotides listed in Table 70 targeting ApoC-III andoligonucleotides in Table 106 targeting Apo(a) were tested in anultra-filtration assay in order to assess plasma protein binding.

TABLE 106 Modified oligonucleotides targeting Apo(a) ISIS GalNAc₃ SEQNo. Sequences (5′ to 3′) Cluster CM ID No 494372 T_(es)G_(es)^(m)C_(es)T_(es) ^(m)C_(es)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(es)G_(es)T_(es) n/a n/a 277 T_(es) ^(m)C_(e) 693401T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es) n/a n/a 277 T_(es) ^(m)C_(e) 681251GalNAc ₃ -7 _(a) - _(o′)T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds)GalNAc₃-7_(a) PO 277 T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681257GalNAc ₃-7 _(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds)GalNAc₃-7_(a) PO 277 T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e)See the Example 74 for table legend. The structure of GalNAc₃-7a wasshown previously in Example 48.

Ultrafree-MC ultrafiltration units (30,000 NMWL, low-binding regeneratedcellulose membrane, Millipore, Bedford, Mass.) were pre-conditioned with300 μL of 0.5% Tween 80 and centrifuged at 2000 g for 10 minutes, thenwith 3004 of a 300 μg/mL solution of a control oligonucleotide in H₂Oand centrifuged at 2000 g for 16 minutes. In order to assessnon-specific binding to the filters of each test oligonucleotide fromTables 70 and 106 to be used in the studies, 300 μL of a 250 ng/mLsolution of oligonucleotide in H₂O at pH 7.4 was placed in thepre-conditioned filters and centrifuged at 2000 g for 16 minutes. Theunfiltered and filtered samples were analyzed by an ELISA assay todetermine the oligonucleotide concentrations. Three replicates were usedto obtain an average concentration for each sample. The averageconcentration of the filtered sample relative to the unfiltered sampleis used to determine the percent of oligonucleotide that is recoveredthrough the filter in the absence of plasma (% recovery).

Frozen whole plasma samples collected in K3-EDTA from normal, drug-freehuman volunteers, cynomolgus monkeys, and CD-1 mice, were purchased fromBioreclamation LLC (Westbury, N.Y.). The test oligonucleotides wereadded to 1.2 mL aliquots of plasma at two concentrations (5 and 150μg/mL). An aliquot (300 μL) of each spiked plasma sample was placed in apre-conditioned filter unit and incubated at 37° C. for 30 minutes,immediately followed by centrifugation at 2000 g for 16 minutes.Aliquots of filtered and unfiltered spiked plasma samples were analyzedby an ELISA to determine the oligonucleotide concentration in eachsample. Three replicates per concentration were used to determine theaverage percentage of bound and unbound oligonucleotide in each sample.The average concentration of the filtered sample relative to theconcentration of the unfiltered sample is used to determine the percentof oligonucleotide in the plasma that is not bound to plasma proteins (%unbound). The final unbound oligonucleotide values are corrected fornon-specific binding by dividing the % unbound by the % recovery foreach oligonucleotide. The final % bound oligonucleotide values aredetermined by subtracting the final % unbound values from 100. Theresults are shown in Table 107 for the two concentrations ofoligonucleotide tested (5 and 150 μg/mL) in each species of plasma. Theresults show that GalNAc conjugate groups do not have a significantimpact on plasma protein binding. Furthermore, oligonucleotides withfull PS internucleoside linkages and mixed PO/PS linkages both bindplasma proteins, and those with full PS linkages bind plasma proteins toa somewhat greater extent than those with mixed PO/PS linkages.

TABLE 107 Percent of modified oligonucleotide bound to plasma proteinsHuman plasma Monkey plasma Mouse plasma ISIS 5 150 5 150 5 150 No. μg/mLμg/mL μg/mL μg/mL μg/mL μg/mL 304801 99.2 98.0 99.8 99.5 98.1 97.2663083 97.8 90.9 99.3 99.3 96.5 93.0 674450 96.2 97.0 98.6 94.4 94.689.3 494372 94.1 89.3 98.9 97.5 97.2 93.6 693401 93.6 89.9 96.7 92.094.6 90.2 681251 95.4 93.9 99.1 98.2 97.8 96.1 681257 93.4 90.5 97.693.7 95.6 92.7

Example 97: Modified Oligonucleotides Targeting TTR Comprising a GalNAc₃Conjugate Group

The oligonucleotides shown in Table 108 comprising a GalNAc conjugatewere designed to target TTR.

TABLE 108 Modified oligonucleotides targeting TTR GalNAc₃ SEQ IDISIS No. Sequences (5′ to 3′) Cluster CM No 666941 GalNAc ₃ -3 _(a-o′) A_(do )T_(es )^(m)C_(es )T_(es )T_(es )G_(es )G_(ds )T_(ds )T_(ds )A_(ds )^(m)C_(ds )A_(ds) GalNAc₃-3 A_(d) 269T_(ds )G_(ds )A_(ds )A_(ds )A_(es )T_(es ) ^(m)C_(es ) ^(m)C_(es )^(m)C_(e) 666942 T_(es )^(m)C_(eo )T_(eo )T_(eo )G_(eo )G_(ds )T_(ds )T_(ds )A_(ds )^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds )A_(ds) GalNAc₃-1 A_(d) 266A_(eo )T_(eo ) ^(m)C_(es ) ^(m)C_(es ) ^(m)C_(eo ) A _(do′) -GalNAc ₃-3_(a) 682876 GalNAc ₃ -3 _(a-o′)T_(es )^(m)C_(es )T_(es )T_(es )G_(es )G_(ds )T_(ds )T_(ds )A_(ds )^(m)C_(ds )A_(ds )T_(ds) GalNAc₃-3 PO 265G_(ds )A_(ds )A_(ds )A_(es )T_(es ) ^(m)C_(es ) ^(m)C_(es ) ^(m)C_(e)682877 GalNAc ₃ -7 _(a-o′)T_(es )^(m)C_(es )T_(es )T_(es )G_(es )G_(ds )T_(ds )T_(ds )A_(ds )^(m)C_(ds )A_(ds )T_(ds) GalNAc₃-7 PO 265G_(ds )A_(ds )A_(ds )A_(es )T_(es ) ^(m)C_(es ) ^(m)C_(es ) ^(m)C_(e)682878 GalNAc ₃ -10 _(a-o′)T_(es )^(m)C_(es )T_(es )T_(es )G_(es )G_(ds )T_(ds )T_(ds )A_(ds )^(m)C_(ds )A_(ds )T_(ds) GalNAc₃-10 PO 265G_(ds )A_(ds )A_(ds )A_(es )T_(es ) ^(m)C_(es ) ^(m)C_(es ) ^(m)C_(e)682879 GalNAc ₃ -13 _(a-o′)T_(es )^(m)C_(es )T_(es )T_(es )G_(es )G_(ds )T_(ds )T_(ds )A_(ds )^(m)C_(ds )A_(ds )T_(ds) GalNAc₃-13 PO 265G_(ds )A_(ds )A_(ds )A_(es )T_(es ) ^(m)C_(es ) ^(m)C_(es ) ^(m)C_(e)682880 GalNAc ₃ -7 _(a-o′)A_(do )T_(es )^(m)C_(es )T_(es )T_(es )G_(es )G_(ds )T_(ds )T_(ds )A_(ds )^(m)C_(ds )A_(ds) GalNAc₃-7 A_(d) 269T_(ds )G_(ds )A_(ds )A_(ds )A_(es )T_(es ) ^(m)C_(es ) ^(m)C_(es )^(m)C_(e) 682881 GalNAc ₃ -10 _(a-o′)A_(do )T_(es )^(m)C_(es )T_(es )T_(es )G_(es )G_(ds )T_(ds )T_(ds )A_(ds ) ^(m)C_(ds)GalNAc₃-10 A_(d) 269 A_(ds )T_(ds )G_(ds )A_(ds )A_(ds )A_(es )T_(es )^(m)C_(es ) ^(m)C_(es ) ^(m)C_(e) 682882 GalNAc ₃ -13_(a-o′)A_(do )T_(es )^(m)C_(es )T_(es )T_(es )G_(es )G_(ds )T_(ds )T_(ds )A_(ds ) ^(m)C_(ds)GalNAc₃-13 A_(d) 269 A_(ds )T_(ds )G_(ds )A_(ds )A_(ds )A_(es )T_(es )^(m)C_(es ) ^(m)C_(es ) ^(m)C_(e) 684056 T_(es )^(m)C_(es )T_(es )T_(es )G_(es )G_(ds )T_(ds )T_(ds )A_(ds )^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds )A_(ds )A_(es) GalNAc₃-19 A_(d)266 T_(es ) ^(m)C_(es ) ^(m)C_(es ) ^(m)C_(eo ) A _(do′) -GalNAc ₃ -19_(a)The legend for Table 108 can be found in Example 74. The structure ofGalNAc₃-1 was shown in Example 9. The structure of GalNAc₃-3_(a) wasshown in Example 39. The structure of GalNAc₃-7_(a) was shown in Example48. The structure of GalNAc₃-10_(a) was shown in Example 46. Thestructure of GalNAc₃-13_(a) was shown in Example 62. The structure ofGalNAc₃-19_(a) was shown in Example 70.

Example 98: Evaluation of Pro-Inflammatory Effects of OligonucleotidesComprising a GalNAc Conjugate in hPMBC Assay

The oligonucleotides listed in Table 109 and were tested forpro-inflammatory effects in an hPMBC assay as described in Examples 23and 24. (See Tables 30, 83, 95, and 108 for descriptions of theoligonucleotides.) ISIS 353512 is a high responder used as a positivecontrol, and the other oligonucleotides are described in Tables 83, 95,and 108. The results shown in Table 109 were obtained using blood fromone volunteer donor. The results show that the oligonucleotidescomprising mixed PO/PS internucleoside linkages produced significantlylower pro-inflammatory responses compared to the same oligonucleotideshaving full PS linkages. Furthermore, the GalNAc conjugate group did nothave a significant effect in this assay.

TABLE 109 ISIS No. E_(max)/EC₅₀ GalNAc₃ cluster Linkages CM 353512 3630n/a PS n/a 420915 802 n/a PS n/a 682881 1311 GalNAc₃-10 PS A_(d) 6828880.26 GalNAc₃-10 PO/PS A_(d) 684057 1.03 GalNAc₃-19 PO/PS A_(d)

Example 99: Binding Affinities of Oligonucleotides Comprising a GalNAcConjugate for the Asialoglycoprotein Receptor

The binding affinities of the oligonucleotides listed in Table 110 (seeTable 76 for descriptions of the oligonucleotides) for theasialoglycoprotein receptor were tested in a competitive receptorbinding assay. The competitor ligand, α1-acid glycoprotein (AGP), wasincubated in 50 mM sodium acetate buffer (pH 5) with 1 Uneuraminidase-agarose for 16 hours at 3TC, and >90% desilylation wasconfirmed by either sialic acid assay or size exclusion chromatography(SEC). Iodine monochloride was used to iodinate the AGP according to theprocedure by Atsma et al. (see J Lipid Res. 1991 January; 32(1):173-81.)In this method, desilylated α1-acid glycoprotein (de-AGP) was added to10 mM iodine chloride, Na¹²⁵I, and 1 M glycine in 0.25 M NaOH. Afterincubation for 10 minutes at room temperature, ¹²⁵I-labeled de-AGP wasseparated from free ¹²⁵I by concentrating the mixture twice utilizing a3 KDMWCO spin column. The protein was tested for labeling efficiency andpurity on a HPLC system equipped with an Agilent SEC-3 column (7.8×300mm) and a ß-RAM counter. Competition experiments utilizing ¹²⁵I-labeledde-AGP and various GalNAc-cluster containing ASOs were performed asfollows. Human HepG2 cells (10⁶ cells/ml) were plated on 6-well platesin 2 ml of appropriate growth media. MEM media supplemented with 10%fetal bovine serum (FBS), 2 mM L-Glutamine and 10 mM HEPES was used.Cells were incubated 16-20 hours @ 3TC with 5% and 10% CO₂ respectively.Cells were washed with media without FBS prior to the experiment. Cellswere incubated for 30 min @37° C. with 1 ml competition mix containingappropriate growth media with 2% FBS, 10⁻⁸ M ¹²⁵I-labeled de-AGP andGalNAc-cluster containing ASOs at concentrations ranging from 10⁻¹¹ to10⁻⁵ M. Non-specific binding was determined in the presence of 10⁻² MGalNAc sugar. Cells were washed twice with media without FBS to removeunbound ¹²⁵I-labeled de-AGP and competitor GalNAc ASO. Cells were lysedusing Qiagen's RLT buffer containing 1% ß-mercaptoethanol. Lysates weretransferred to round bottom assay tubes after a brief 10 min freeze/thawcycle and assayed on a γ-counter. Non-specific binding was subtractedbefore dividing ¹²⁵I protein counts by the value of the lowestGalNAc-ASO concentration counts. The inhibition curves were fittedaccording to a single site competition binding equation using anonlinear regression algorithm to calculate the binding affinities(K_(D)'s).

The results in Table 110 were obtained from experiments performed onfive different days. Results for oligonucleotides marked withsuperscript “a” are the average of experiments run on two differentdays. The results show that the oligonucleotides comprising a GalNAcconjugate group on the 5′-end bound the asialoglycoprotein receptor onhuman HepG2 cells with 1.5 to 16-fold greater affinity than theoligonucleotides comprising a GalNAc conjugate group on the 3′-end.

TABLE 110 Asialoglycoprotein receptor binding assay resultsOligonucleotide end to which GalNAc ISIS No. GalNAc conjugate conjugateis attached K_(D) (nM) 661161^(a) GalNAc₃-3  5′ 3.7 666881^(a)GalNAc₃-10 5′ 7.6 666981  GalNAc₃-7  5′ 6.0 670061  GalNAc₃-13 5′ 7.4655861^(a) GalNAc₃-1  3′ 11.6 677841^(a) GalNAc₃-19 3′ 60.8

Example 100: Antisense Inhibition In Vivo by Oligonucleotides Comprisinga GalNAc Conjugate Group Targeting Apo(a) In Vivo

The oligonucleotides listed in Table 111a below were tested in a singledose study for duration of action in mice.

TABLE 111a Modified ASOs targeting APO(a) ISIS GalNAc₃ SEQ No.Sequences (5′ to 3′) Cluster CM ID No. 681251 GalNAc ₃ -7 _(a) -_(o′)T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 277 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681257 GalNAc ₃ -7_(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 277 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e)The structure of GalNAc₃-7_(a) was shown in Example 48.

Treatment

Female transgenic mice that express human Apo(a) were each injectedsubcutaneously once per week, for a total of 6 doses, with anoligonucleotide and dosage listed in Table 111b or with PBS. Eachtreatment group consisted of 3 animals. Blood was drawn the day beforedosing to determine baseline levels of Apo(a) protein in plasma and at72 hours, 1 week, and 2 weeks following the first dose. Additional blooddraws will occur at 3 weeks, 4 weeks, 5 weeks, and 6 weeks following thefirst dose. Plasma Apo(a) protein levels were measured using an ELISA.The results in Table 111b are presented as the average percent of plasmaApo(a) protein levels for each treatment group, normalized to baselinelevels (% BL), The results show that the oligonucleotides comprising aGalNAc conjugate group exhibited potent reduction in Apo(a) expression.This potent effect was observed for the oligonucleotide that comprisesfull PS internucleoside linkages and the oligonucleotide that comprisesmixed PO and PS linkages.

TABLE 111b Apo(a) plasma protein levels Apo(a) at Apo(a) at Apo(a) atDosage 72 hours 1 week 3 weeks ISIS No. (mg/kg) (% BL) (% BL) (% BL) PBSn/a 116 104 107 681251 0.3 97 108 93 1.0 85 77 57 3.0 54 49 11 10.0 2315 4 681257 0.3 114 138 104 1.0 91 98 54 3.0 69 40 6 10.0 30 21 4

Example 101: Antisense Inhibition by Oligonucleotides Comprising aGalNAc Cluster Linked Via a Stable Moiety

The oligonucleotides listed in Table 112 were tested for inhibition ofmouse APOC-III expression in vivo. C57Bl/6 mice were each injectedsubcutaneously once with an oligonucleotide listed in Table 112 or withPBS. Each treatment group consisted of 4 animals. Each mouse treatedwith ISIS 440670 received a dose of 2, 6, 20, or 60 mg/kg. Each mousetreated with ISIS 680772 or 696847 received 0.6, 2, 6, or 20 mg/kg. TheGalNAc conjugate group of ISIS 696847 is linked via a stable moiety, aphosphorothioate linkage instead of a readily cleavable phosphodiestercontaining linkage. The animals were sacrificed 72 hours after the dose.Liver APOC-III mRNA levels were measured using real-time PCR. APOC-IIImRNA levels were normalized to cyclophilin mRNA levels according tostandard protocols. The results are presented in Table 112 as theaverage percent of APOC-III mRNA levels for each treatment grouprelative to the saline control group. The results show that theoligonucleotides comprising a GalNAc conjugate group were significantlymore potent than the oligonucleotide lacking a conjugate group.Furthermore, the oligonucleotide comprising a GalNAc conjugate grouplinked to the oligonucleotide via a cleavable moiety (ISIS 680772) waseven more potent than the oligonucleotide comprising a GalNAc conjugategroup linked to the oligonucleotide via a stable moiety (ISIS 696847).

TABLE 112 Modified oligonucleotides targeting mouse APOC-III APOC-IIIISIS Dosage mRNA (% SEQ No. Sequences (5′ to 3′) CM (mg/kg) PBS) ID No.440670 ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds)T_(ds)A_(ds) n/a 2 92 271G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es)A_(es)G_(es) ^(m)C_(es)A_(e) 6 86 2059 60 37 680772 GalNAc ₃ -7 _(a-o′) ^(m)C_(es)A_(es)G_(es)^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds) PO 0.6 79 271T_(ds)T_(ds)A_(ds)G_(ds )G_(ds)G_(ds)A_(ds) ^(m)C_(es )A_(es)G_(es)^(m)C_(es)A_(e) 2 58 6 31 20 13 696847 GalNAc ₃ -7 _(a-s′)^(m)C_(es)A_(es)G_(es) ^(m)C_(es)T_(es)T_(ds)T_(ds)A_(ds)T_(ds) n/a (PS)0.6 83 271 T_(ds)A_(ds)G_(ds)G_(ds)G_(ds)A_(ds) ^(m)C_(es )A_(es)G_(es)^(m)C_(es)A_(e) 2 73 6 40 20 28The structure of GalNAc₃-7_(a) was shown in Example 48.

Example 102: Distribution in Liver of Antisense OligonucleotidesComprising a GalNAc Conjugate

The liver distribution of ISIS 353382 (see Table 36) that does notcomprise a GalNAc conjugate and ISIS 655861 (see Table 36) that doescomprise a GalNAc conjugate was evaluated. Male balb/c mice weresubcutaneously injected once with ISIS 353382 or 655861 at a dosagelisted in Table 113. Each treatment group consisted of 3 animals exceptfor the 18 mg/kg group for ISIS 655861, which consisted of 2 animals.The animals were sacrificed 48 hours following the dose to determine theliver distribution of the oligonucleotides. In order to measure thenumber of antisense oligonucleotide molecules per cell, a Ruthenium (II)tris-bipyridine tag (MSD TAG, Meso Scale Discovery) was conjugated to anoligonucleotide probe used to detect the antisense oligonucleotides. Theresults presented in Table 113 are the average concentrations ofoligonucleotide for each treatment group in units of millions ofoligonucleotide molecules per cell. The results show that at equivalentdoses, the oligonucleotide comprising a GalNAc conjugate was present athigher concentrations in the total liver and in hepatocytes than theoligonucleotide that does not comprise a GalNAc conjugate. Furthermore,the oligonucleotide comprising a GalNAc conjugate was present at lowerconcentrations in non-parenchymal liver cells than the oligonucleotidethat does not comprise a GalNAc conjugate. And while the concentrationsof ISIS 655861 in hepatocytes and non-parenchymal liver cells weresimilar per cell, the liver is approximately 80% hepatocytes by volume.Thus, the majority of the ISIS 655861 oligonucleotide that was presentin the liver was found in hepatocytes, whereas the majority of the ISIS353382 oligonucleotide that was present in the liver was found innon-parenchymal liver cells.

TABLE 113 Concentration in Concentration Concentration non-parenchymalin whole liver in hepatocytes liver cells ISIS Dosage (molecules*(molecules* (molecules* No. (mg/kg) 10{circumflex over ( )}6 per cell)10{circumflex over ( )}6 per cell) 10{circumflex over ( )}6 per cell)353382 3 9.7 1.2 37.2 10 17.3 4.5 34.0 20 23.6 6.6 65.6 30 29.1 11.780.0 60 73.4 14.8 98.0 90 89.6 18.5 119.9 655861 0.5 2.6 2.9 3.2 1 6.27.0 8.8 3 19.1 25.1 28.5 6 44.1 48.7 55.0 18 76.6 82.3 77.1

Example 103: Duration of Action In Vivo of Oligonucleotides TargetingAPOC-III Comprising a GalNAc₃ Conjugate

The oligonucleotides listed in Table 114 below were tested in a singledose study for duration of action in mice.

TABLE 114 Modified ASOs targeting APOC-III ISIS GalNAc₃ SEQ No.Sequences (5′ to 3′) Cluster CM ID No. 304801 A_(es)G_(es)^(m)C_(es)T_(es)T_(es) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(es)T_(es) n/a n/a 244T_(es)A_(es)T_(e) 663084 GalNAc ₃ -3 _(a) - _(o′) A _(do)A_(es)G_(eo)^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds)GalNAc₃-3a A_(d) 260 ^(m)C_(ds)A_(ds)G_(ds)^(m)C_(ds)T_(eo)T_(eo)T_(es)A_(es)T_(e) 679241 A_(es)G_(eo)^(m)C_(eo)T_(eo)T_(eo) ^(m)C_(ds)T_(ds)T_(ds)G_(ds)T_(ds) ^(m)C_(ds)^(m)C_(ds)A_(ds)G_(ds) ^(m)C_(ds)T_(eo)T_(eo) GalNAc₃-19a A_(d) 245T_(es)A_(es)T_(eo) A _(do′) -GalNAc ₃ -19 _(a)The structure of GalNAc₃-3_(a) was shown in Example 39, andGalNAc₃-19_(a) was shown in Example 70.

Treatment

Female transgenic mice that express human APOC-III were each injectedsubcutaneously once with an oligonucleotide listed in Table 114 or withPBS. Each treatment group consisted of 3 animals. Blood was drawn beforedosing to determine baseline and at 3, 7, 14, 21, 28, 35, and 42 daysfollowing the dose. Plasma triglyceride and APOC-III protein levels weremeasured as described in Example 20. The results in Table 115 arepresented as the average percent of plasma triglyceride and APOC-IIIlevels for each treatment group, normalized to baseline levels. Acomparison of the results in Table 71 of example 79 with the results inTable 115 below show that oligonucleotides comprising a mixture ofphosphodiester and phosphorothioate internucleoside linkages exhibitedincreased duration of action than equivalent oligonucleotides comprisingonly phosphorothioate internucleoside linkages.

TABLE 115 Plasma triglyceride and APOC-III protein levels in transgenicmice Time point Triglyc- APOC-III (days erides protein ISIS Dosage post-(% (% GalNAc₃ No. (mg/kg) dose) baseline) baseline) Cluster CM PBS n/a 396 101 n/a n/a 7 88 98 14 91 103 21 69 92 28 83 81 35 65 86 42 72 88304801 30 3 42 46 n/a n/a 7 42 51 14 59 69 21 67 81 28 79 76 35 72 95 4282 92 663084 10 3 35 28 GalNAc₃-3a A_(d) 7 23 24 14 23 26 21 23 29 28 3022 35 32 36 42 37 47 679241 10 3 38 30 GalNAc₃-19a A_(d) 7 31 28 14 3022 21 36 34 28 48 34 35 50 45 42 72 64

Example 104: Synthesis of Oligonucleotides Comprising a 5′-GalNAc₂Conjugate

Compound 120 is commercially available, and the synthesis of compound126 is described in Example 49. Compound 120 (1 g, 2.89 mmol), HBTU(0.39 g, 2.89 mmol), and HOBt (1.64 g, 4.33 mmol) were dissolved in DMF(10 mL. and N,N-diisopropylethylamine (1.75 mL, 10.1 mmol) were added.After about 5 min, aminohexanoic acid benzyl ester (1.36 g, 3.46 mmol)was added to the reaction. After 3 h, the reaction mixture was pouredinto 100 mL of 1 M NaHSO₄ and extracted with 2×50 mL ethyl acetate.Organic layers were combined and washed with 3×40 mL sat NaHCO₃ and2×brine, dried with Na₂SO₄, filtered and concentrated. The product waspurified by silica gel column chromatography (DCM:EA:Hex, 1:1:1) toyield compound 231. LCMS and NMR were consistent with the structure.Compounds 231 (1.34 g, 2.438 mmol) was dissolved in dichloromethane (10mL) and trifluoracetic acid (10 mL) was added. After stirring at roomtemperature for 2 h, the reaction mixture was concentrated under reducedpressure and co-evaporated with toluene (3×10 mL). The residue was driedunder reduced pressure to yield compound 232 as the trifluoracetatesalt. The synthesis of compound 166 is described in Example 54. Compound166 (3.39 g, 5.40 mmol) was dissolved in DMF (3 mL). A solution ofcompound 232 (1.3 g, 2.25 mmol) was dissolved in DMF (3 mL) andN,N-diisopropylethylamine (1.55 mL) was added. The reaction was stirredat room temperature for 30 minutes, then poured into water (80 mL) andthe aqueous layer was extracted with EtOAc (2×100 mL). The organic phasewas separated and washed with sat. aqueous NaHCO₃ (3×80 mL), 1 M NaHSO₄(3×80 mL) and brine (2×80 mL), then dried (Na₂SO₄), filtered, andconcentrated. The residue was purified by silica gel columnchromatography to yield compound 233. LCMS and NMR were consistent withthe structure. Compound 233 (0.59 g, 0.48 mmol) was dissolved inmethanol (2.2 mL) and ethyl acetate (2.2 mL). Palladium on carbon (10 wt% Pd/C, wet, 0.07 g) was added, and the reaction mixture was stirredunder hydrogen atmosphere for 3 h. The reaction mixture was filteredthrough a pad of Celite and concentrated to yield the carboxylic acid.The carboxylic acid (1.32 g, 1.15 mmol, cluster free acid) was dissolvedin DMF (3.2 mL). To this N,N-diisopropylethylamine (0.3 mL, 1.73 mmol)and PFPTFA (0.30 mL, 1.73 mmol) were added. After 30 min stirring atroom temperature the reaction mixture was poured into water (40 mL) andextracted with EtOAc (2×50 mL). A standard work-up was completed asdescribed above to yield compound 234. LCMS and NMR were consistent withthe structure. Oligonucleotide 235 was prepared using the generalprocedure described in Example 46. The GalNAc₂ cluster portion(GalNAc₂-24_(a)) of the conjugate group GalNAc2-24 can be combined withany cleavable moiety present on the oligonucleotide to provide a varietyof conjugate groups. The structure of GalNAc2-24 (GalNAc2-24_(a)-CM) isshown below:

Example 105: Synthesis of Oligonucleotides Comprising a GalNAc₁-25Conjugate

The synthesis of compound 166 is described in Example 54.Oligonucleotide 236 was prepared using the general procedure describedin Example 46.

Alternatively, oligonucleotide 236 was synthesized using the schemeshown below, and compound 238 was used to form the oligonucleotide 236using procedures described in Example 10.

The GalNAc₁ cluster portion (GalNAc₁-25_(a)) of the conjugate groupGalNAc₁-25 can be combined with any cleavable moiety present on theoligonucleotide to provide a variety of conjugate groups. The structureof GalNAc1-25 (GalNAc₁-25_(a)-CM) is shown below:

Example 106: Antisense Inhibition In Vivo by Oligonucleotides TargetingSRB-1 Comprising a 5′-GalNAc₂ or a 5′-GalNAc₃ Conjugate

Oligonucleotides listed in Tables 116 and 117 were tested indose-dependent studies for antisense inhibition of SRB-1 in mice.

Treatment

Six to week old, male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.)were injected subcutaneously once with 2, 7, or 20 mg/kg of ISIS No.440762; or with 0.2, 0.6, 2, 6, or 20 mg/kg of ISIS No. 686221, 686222,or 708561; or with saline. Each treatment group consisted of 4 animals.The mice were sacrificed 72 hours following the final administration.Liver SRB-1 mRNA levels were measured using real-time PCR. SRB-1 mRNAlevels were normalized to cyclophilin mRNA levels according to standardprotocols. The antisense oligonucleotides lowered SRB-1 mRNA levels in adose-dependent manner, and the ED₅₀ results are presented in Tables 116and 117. Although previous studies showed that trivalentGalNAc-conjugated oligonucleotides were significantly more potent thandivalent GalNAc-conjugated oligonucleotides, which were in turnsignificantly more potent than monovalent GalNAc conjugatedoligonucleotides (see, e.g., Khorev et al., Bioorg. & Med. Chem., Vol.16, 5216-5231 (2008)), treatment with antisense oligonucleotidescomprising monovalent, divalent, and trivalent GalNAc clusters loweredSRB-1 mRNA levels with similar potencies as shown in Tables 116 and 117.

TABLE 116 Modified oligonucleotides targeting SRB-1 ISIS ED₅₀ SEQ No.Sequences (5′ to 3′) GalNAc Cluster (mg/kg) ID No 440762 T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) n/a 4.7 246 686221 GalNAc ₂ -24 _(a) -_(o′) A _(do)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₂-24_(a) 0.39 250^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) 686222 GalNAc ₃ -13 _(a) - _(o′) A_(do)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₃-13_(a) 0.41 250^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k)See Example 93 for table legend. The structure of GalNAc₃-13a was shownin Example 62, and the structure of GalNAc₂-24a was shown in Example104.

TABLE 117 Modified oligonucleotides targeting SRB-1 ISIS ED₅₀ SEQ No.Sequences (5′ to 3′) GalNAc Cluster (mg/kg) ID No 440762 T_(ks)^(m)C_(ks)A_(ds)G_(ds)T_(ds) ^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds)^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k) n/a 5 246 708561 GalNAc ₁ -25 _(a) -_(o′)T_(ks) ^(m)C_(ks)A_(ds)G_(ds)T_(ds)^(m)C_(ds)A_(ds)T_(ds)G_(ds)A_(ds) GalNAc₁-25_(a) 0.4 246^(m)C_(ds)T_(ds)T_(ks) ^(m)C_(k)See Example 93 for table legend. The structure of GalNAc₁-25a was shownin Example 105.

The concentrations of the oligonucleotides in Tables 116 and 117 inliver were also assessed, using procedures described in Example 75. Theresults shown in Tables 117a and 117b below are the average totalantisense oligonucleotide tissues levels for each treatment group, asmeasured by UV in units of μg oligonucleotide per gram of liver tissue.The results show that the oligonucleotides comprising a GalNAc conjugategroup accumulated in the liver at significantly higher levels than thesame dose of the oligonucleotide lacking a GalNAc conjugate group.Furthermore, the antisense oligonucleotides comprising one, two, orthree GalNAc ligands in their respective conjugate groups allaccumulated in the liver at similar levels. This result is surprising inview of the Khorev et al. literature reference cited above and isconsistent with the activity data shown in Tables 116 and 117 above.

TABLE 117a Liver concentrations of oligonucleotides comprising a GalNAc₂or GalNAc₃ conjugate group [Antisense Dosage oligonucleotide] ISIS No.(mg/kg) (μg/g) GalNAc cluster CM 440762 2 2.1 n/a n/a 7 13.1 20 31.1686221 0.2 0.9 GalNAc₂-24_(a) A_(d) 0.6 2.7 2 12.0 6 26.5 686222 0.2 0.5GalNAc₃-13_(a) A_(d) 0.6 1.6 2 11.6 6 19.8

TABLE 117b Liver concentrations of oligonucleotides comprising a GalNAc₁conjugate group [Antisense Dosage oligonucleotide] ISIS No. (mg/kg)(μg/g) GalNAc cluster CM 440762 2 2.3 n/a n/a 7 8.9 20 23.7 708561 0.20.4 GalNAc₁-25_(a) PO 0.6 1.1 2 5.9 6 23.7 20 53.9

Example 107: Synthesis of Oligonucleotides Comprising a GalNAc₁-26 orGalNAc₁-27 Conjugate

Oligonucleotide 239 is synthesized via coupling of compound 47 (seeExample 15) to acid 64 (see Example 32) using HBTU and DIEA in DMF. Theresulting amide containing compound is phosphitylated, then added to the5′-end of an oligonucleotide using procedures described in Example 10.The GalNAc₁ cluster portion (GalNAc₁-26_(a)) of the conjugate groupGalNAc₁-26 can be combined with any cleavable moiety present on theoligonucleotide to provide a variety of conjugate groups. The structureof GalNAc₁-26 (GalNAc₁-26_(a)-CM) is shown below:

In order to add the GalNAc₁ conjugate group to the 3′-end of anoligonucleotide, the amide formed from the reaction of compounds 47 and64 is added to a solid support using procedures described in Example 7.The oligonucleotide synthesis is then completed using proceduresdescribed in Example 9 in order to form oligonucleotide 240.

The GalNAc₁ cluster portion (GalNAc₁-27_(a)) of the conjugate groupGalNAc₁-27 can be combined with any cleavable moiety present on theoligonucleotide to provide a variety of conjugate groups. The structureof GalNAc₁-27 (GalNAc₁-27_(a)-CM) is shown below:

Example 108: Antisense Inhibition In Vivo by Oligonucleotides Comprisinga GalNAc Conjugate Group Targeting Apo(a) In Vivo

The oligonucleotides listed in Table 118 below were tested in a singledose study in mice.

TABLE 118 Modified ASOs targeting APO(a) ISIS GalNAc₃ SEQ No.Sequences (5′ to 3′) Cluster CM ID No. 494372 T_(es)G_(es)^(m)C_(es)T_(es) ^(m)C_(es)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds) ^(m)C_(ds) n/a n/a277 T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681251 GalNAc ₃ -7 _(a) -_(o′)T_(es)G_(es) ^(m)C_(es)T_(es) ^(m)C_(es)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 277 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(es)G_(es)T_(es)T_(es) ^(m)C_(e) 681255 GalNAc ₃ -3_(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-3a PO 277 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) 681256 GalNAc ₃ -10_(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-10a PO 277 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) 681257 GalNAc ₃ -7_(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-7a PO 277 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) 681258 GalNAc ₃ -13_(a) - _(o′)T_(es)G_(eo) ^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds) GalNAc₃-13a PO 277 T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo)T_(es)T_(es) ^(m)C_(e) 681260 T_(es)G_(eo)^(m)C_(eo)T_(eo) ^(m)C_(eo)^(m)C_(ds)G_(ds)T_(ds)T_(ds)G_(ds)G_(ds)T_(ds)G_(ds)^(m)C_(ds)T_(ds)T_(eo)G_(eo) GalNAc₃-19a A_(d) 276 T_(es)T_(es)^(m)C_(eo) A _(do′) -GalNAc ₃ -19The structure of GalNAc₃-7_(a) was shown in Example 48.

Treatment

Male transgenic mice that express human Apo(a) were each injectedsubcutaneously once with an oligonucleotide and dosage listed in Table119 or with PBS. Each treatment group consisted of 4 animals. Blood wasdrawn the day before dosing to determine baseline levels of Apo(a)protein in plasma and at 1 week following the first dose. Additionalblood draws will occur weekly for approximately 8 weeks. Plasma Apo(a)protein levels were measured using an ELISA. The results in Table 119are presented as the average percent of plasma Apo(a) protein levels foreach treatment group, normalized to baseline levels (% BL), The resultsshow that the antisense oligonucleotides reduced Apo(a) proteinexpression. Furthermore, the oligonucleotides comprising a GalNAcconjugate group exhibited even more potent reduction in Apo(a)expression than the oligonucleotide that does not comprise a conjugategroup.

TABLE 119 Apo(a) plasma protein levels Apo(a) at 1 week ISIS No. Dosage(mg/kg) (% BL) PBS n/a 143 494372 50 58 681251 10 15 681255 10 14 68125610 17 681257 10 24 681258 10 22 681260 10 26

Example 109: Synthesis of Oligonucleotides Comprising a GalNAc₁-28 orGalNAc₁-29 Conjugate

Oligonucleotide 241 is synthesized using procedures similar to thosedescribed in Example 71 to form the phosphoramidite intermediate,followed by procedures described in Example 10 to synthesize theoligonucleotide. The GalNAc₁ cluster portion (GalNAc₁-28_(a)) of theconjugate group GalNAc₁-28 can be

combined with any cleavable moiety present on the oligonucleotide toprovide a variety of conjugate groups. The structure of GalNAc₁-28(GalNAc₁-28_(a)-CM) is shown below:

In order to add the GalNAc₁ conjugate group to the 3′-end of anoligonucleotide, procedures similar to those described in Example 71 areused to form the hydroxyl intermediate, which is then added to the solidsupport using procedures described in Example 7. The oligonucleotidesynthesis is then completed using procedures described in Example 9 inorder to form oligonucleotide 242.

The GalNAc₁ cluster portion (GalNAc₁-29_(a)) of the conjugate groupGalNAc₁-29 can be combined with any cleavable moiety present on theoligonucleotide to provide a variety of conjugate groups. The structureof GalNAc₁-29 (GalNAc₁-29_(a)-CM) is shown below:

Example 110: Synthesis of Oligonucleotides Comprising a GalNAc₁-30Conjugate

Oligonucleotide 246 comprising a GalNAc₁-30 conjugate group, wherein Yis selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl,amino, substituted amino, azido, alkenyl or alkynyl, is synthesized asshown above. The GalNAc₁ cluster portion (GalNAc₁-30_(a)) of theconjugate group GalNAc₁-30 can be combined with any cleavable moiety toprovide a variety of conjugate groups. In certain embodiments, Y is partof the cleavable moiety. In certain embodiments, Y is part of a stablemoiety, and the cleavable moiety is present on the oligonucleotide. Thestructure of GalNAc₁-30_(a) is shown below:

Example 111: Synthesis of Oligonucleotides Comprising a GalNAc₂-31 orGalNAc₂-32 Conjugate

Oligonucleotide 250 comprising a GalNAc₂-31 conjugate group, wherein Yis selected from O, S, a

substituted or unsubstituted C₁-C₁₀ alkyl, amino, substituted amino,azido, alkenyl or alkynyl, is synthesized as shown above. The GalNAc₂cluster portion (GalNAc₂-31_(a)) of the conjugate group GalNAc₂-31 canbe combined with any cleavable moiety to provide a variety of conjugategroups. In certain embodiments, the Y-containing group directly adjacentto the 5′-end of the oligonucleotide is part of the cleavable moiety. Incertain embodiments, the Y-containing group directly adjacent to the5′-end of the oligonucleotide is part of a stable moiety, and thecleavable moiety is present on the oligonucleotide. The structure ofGalNAc₂-31_(a) is shown below:

The synthesis of an oligonucleotide comprising a GalNAc₂-32 conjugate isshown below.

Oligonucleotide 252 comprising a GalNAc₂-32 conjugate group, wherein Yis selected from O, S, a substituted or unsubstituted C₁-C₁₀ alkyl,amino, substituted amino, azido, alkenyl or alkynyl, is synthesized asshown above. The GalNAc₂ cluster portion (GalNAc₂-32_(a)) of theconjugate group GalNAc₂-32 can be combined with any cleavable moiety toprovide a variety of conjugate groups. In certain embodiments, theY-containing group directly adjacent to the 5′-end of theoligonucleotide is part of the cleavable moiety. In certain embodiments,the Y-containing group directly adjacent to the 5′-end of theoligonucleotide is part of a stable moiety, and the cleavable moiety ispresent on the oligonucleotide. The structure of GalNAc₂-32_(a) is shownbelow:

Example 112: Modified Oligonucleotides Comprising a GalNAc₁ Conjugate

The oligonucleotides in Table 120 targeting SRB-1 were synthesized witha GalNAc₁ conjugate group in order to further test the potency ofoligonucleotides comprising conjugate groups that contain one GalNAcligand.

TABLE 120 GalNAc SEQ ISIS No. Sequence (5′ to 3′) cluster CM ID NO.711461 GalNAc ₁ -25 _(a-o′) A _(do )G_(es ) ^(m)C_(es )T_(es )T_(es )^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds) GalNAc₁-25_(a) A_(d)254 T_(ds )G_(ds )A_(ds ) ^(m)C_(ds )T_(ds )T_(es ) ^(m)C_(es )^(m)C_(es )T_(es )T_(e) 711462 GalNAc ₁ -25 _(a-o′)G_(es )^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds )^(m)C_(ds )A_(ds )T_(ds) GalNAc₁-25_(a) PO 252 G_(ds )A_(ds )^(m)C_(ds )T_(ds )T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(e) 711463GalNAc ₁ -25 _(a-o′)G_(es ) ^(m)C_(eo )T_(eo )T_(eo )^(m)C_(eo )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds) GalNAc₁-25_(a)PO 252 G_(ds )A_(ds ) ^(m)C_(ds )T_(ds )T_(eo ) ^(m)C_(eo )^(m)C_(es )T_(es )T_(e) 711465 GalNAc ₁ -26 _(a-o′) A _(do )G_(es )^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds )^(m)C_(ds )A_(ds) GalNAc₁-26_(a) A_(d) 254 T_(ds )G_(ds )A_(ds )^(m)C_(ds )T_(ds )T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(e) 711466GalNAc ₁ -26 _(a-o′)G_(es ) ^(m)C_(es )T_(es )T_(es )^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds) GalNAc₁-26_(a)PO 252 G_(ds )A_(ds ) ^(m)C_(ds )T_(ds )T_(es ) ^(m)C_(es )^(m)C_(es )T_(es )T_(e) 711467 GalNAc ₁ -26 _(a-o′)G_(es )^(m)C_(eo )T_(eo )T_(eo ) ^(m)C_(eo )A_(ds )G_(ds )T_(ds )^(m)C_(ds )A_(ds )T_(ds) GalNAc₁-26_(a) PO 252 G_(ds )A_(ds )^(m)C_(ds )T_(ds )T_(eo ) ^(m)C_(eo ) ^(m)C_(es )T_(es )T_(e) 711468GalNAc ₁ -28 _(a-o′) A _(do )G_(es ) ^(m)C_(es )T_(es )T_(es )^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds) GalNAc₁-28_(a) A_(d)254 T_(ds )G_(ds )A_(ds ) ^(m)C_(ds )T_(ds )T_(es ) ^(m)C_(es )^(m)C_(es )T_(es )T_(e) 711469 GalNAc ₁ -28 _(a-o′)G_(es )^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds )^(m)C_(ds )A_(ds )T_(ds) GalNAc₁-28_(a) PO 252 G_(ds )A_(ds )^(m)C_(ds )T_(ds )T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(e) 711470GalNAc ₁ -28 _(a-o′)G_(es ) ^(m)C_(eo )T_(eo )T_(eo )^(m)C_(eo )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds) GalNAc₁-28_(a)PO 252 G_(ds )A_(ds ) ^(m)C_(ds )T_(ds )T_(eo ) ^(m)C_(eo )^(m)C_(es )T_(es )T_(e) 713844 G_(es ) ^(m)C_(es )T_(es )T_(es )^(m)C_(es )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds )^(m)C_(ds )T_(ds) GalNAc₁-27_(a) PO 252 T_(es ) ^(m)C_(es )^(m)C_(es )T_(es )T_(eo′-) GalNAc ₁ -27 _(a) 713845 G_(es )^(m)C_(eo )T_(eo )T_(eo ) ^(m)C_(eo )A_(ds )G_(ds )T_(ds )^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds ) ^(m)C_(ds )T_(ds) GalNAc₁-27_(a)PO 252 T_(eo ) ^(m)C_(eo ) ^(m)C_(es )T_(es )T_(eo′-) GalNAc ₁ -27 _(a)713846 G_(es ) ^(m)C_(eo )T_(eo )T_(eo )^(m)C_(eo )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds )^(m)C_(ds )T_(ds) GalNAc₁-27_(a) A_(d) 253 T_(eo ) ^(m)C_(eo )^(m)C_(es )T_(es )T_(eo )A_(do′-) GalNAc ₁ -27 _(a) 713847 G_(es )^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds )^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds ) ^(m)C_(ds )T_(ds) GalNAc₁-29_(a)PO 252 T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(eo′-) GalNAc ₁ -29 _(a)713848 G_(es ) ^(m)C_(eo )T_(eo )T_(eo )^(m)C_(eo )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds )^(m)C_(ds )T_(ds) GalNAc₁-29_(a) PO 252 T_(eo ) ^(m)C_(eo )^(m)C_(es )T_(es )T_(eo′-) GalNAc ₁ -29 _(a) 713849 G_(es )^(m)C_(es )T_(es )T_(es ) ^(m)C_(es )A_(ds )G_(ds )T_(ds )^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds ) ^(m)C_(ds )T_(ds) GalNAc₁-29_(a)A_(d) 253 T_(es ) ^(m)C_(es ) ^(m)C_(es )T_(es )T_(eo ) A _(do′-) GalNAc₁ -29 _(a) 713850 G_(es ) ^(m)C_(eo )T_(eo )T_(eo )^(m)C_(eo )A_(ds )G_(ds )T_(ds ) ^(m)C_(ds )A_(ds )T_(ds )G_(ds )A_(ds )^(m)C_(ds )T_(ds) GalNAc₁-29_(a) A_(d) 253 T_(eo ) ^(m)C_(eo )^(m)C_(es )T_(es )T_(eo ) A _(do′-) GalNAc ₁ -29 _(a)

Example 113: Design and Screening of Duplexed Antisense CompoundsTargeting Apolipoprotein

In accordance with the present invention, a series of nucleic acidduplexes comprising the antisense compounds of the present invention andtheir complements are designed to target apolipoprotein C-III. Thenucleobase sequence of the antisense strand of the duplex comprises atleast a portion of an oligonucleotide in Table 121. The ends of thestrands may be modified by the addition of one or more natural ormodified nucleobases to form an overhang. The sense strand of the dsRNAis then designed and synthesized as the complement of the antisensestrand and may also contain modifications or additions to eitherterminus. For example, in one embodiment, both strands of the dsRNAduplex would be complementary over the central nucleobases, each havingoverhangs at one or both termini.

For example, a duplex comprising an antisense strand having the sequenceCGAGAGGCGGACGGGACCG (SEQ ID NO: 12) and having a two-nucleobase overhangof deoxythymidine (dT) would have the following structure (Antisense SEQID NO: 13, Complement SEQ ID NO: 14):

  cgagaggcggacgggaccgTT Antisense Strand    |||||||||||||||||||(SEQ ID NO: 13) TTgctctccgcctgccctggc Complement  (SEQ ID NO: 14)

In another embodiment, a duplex comprising an antisense strand havingthe same sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 12) may be preparedwith blunt ends (no single stranded overhang) as shown (Antisense SEQ IDNO: 15, Complement SEQ ID NO: 16):

cgagaggcggacgggaccg Antisense Strand  |||||||||||||||||||(SEQ ID NO: 15) gctctccgcctgccctggc Complement  (SEQ ID NO: 16)

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from Dharmacon Research Inc., (Lafayette, Colo.). Oncesynthesized, the complementary strands are annealed. The single strandsare aliquoted and diluted to a concentration of 50 μM. Once diluted, 30μL of each strand is combined with 154 of a 5× solution of annealingbuffer. The final concentration of said buffer is 100 mM potassiumacetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The finalvolume is 75 μL. This solution is incubated for 1 minute at 90° C. andthen centrifuged for 15 seconds. The tube is allowed to sit for 1 hourat 37° C. at which time the dsRNA duplexes are used in experimentation.The final concentration of the dsRNA duplex is 20 μM. This solution canbe stored frozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the duplexed antisense compounds are evaluated for theirability to modulate apolipoprotein C-III expression.

When cells reached 80% confluency, they are treated with duplexedantisense compounds of the invention. For cells grown in 96-well plates,wells are washed once with 200 μL OPTI-MEM-1™ reduced-serum medium(Gibco BRL) and then treated with 130 μL of OPTI-MEM-1™ mediumcontaining 12 μg/mL LIPOFECTIN™ reagent (Gibco BRL) and the desiredduplex antisense compound at a final concentration of 200 nM. After 5hours of treatment, the medium is replaced with fresh medium. Cells areharvested 16 hours after treatment, at which time RNA is isolated andtarget reduction measured by RT-PCR.

Example 114: Antisense Inhibition of Human Apolipoprotein C-IIIExpression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOEWings and a Deoxy Gap

In accordance with the present invention, a series of antisensecompounds was designed to target different regions of the humanapolipoprotein C-III RNA, using published sequences (nucleotides 6238608to 6242565 of GenBank accession number NT_035088.1, representing agenomic sequence, incorporated herein as SEQ ID NO: 3, and GenBankaccession number NM_000040.1, incorporated herein as SEQ ID NO: 1). Thecompounds are shown in Table 121. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe compound binds. All compounds in Table 121 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-O-(2-methoxyethyl) nucleotides, also knownas (2′-MOE) nucleotides. The internucleoside (backbone) linkages arephosphorothioate (P═S) throughout the oligonucleotide. All cytidineresidues are 5-methylcytidines. The compounds were analyzed for theireffect on human apolipoprotein C-III mRNA levels by quantitativereal-time PCR as described in other examples herein. Data are averagesfrom three experiments in which HepG2 cells were treated with theantisense oligonucleotides of the present invention. The positivecontrol for each datapoint is identified in the table by sequence IDnumber. If present, “N. D.” indicates “no data”.

TABLE 121Inhibition of human apolipoprotein C-III mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET SEQ ID TARGET % SEQ CONTROL ISIS # REGION NO SITE SEQUENCE INHIBID NO SEQ ID NO 167824 5′UTR 3 414 ctggagcagctgcctctagg 79 19 1 167835Coding 3 1292 ccctgcatgaagctgagaag 60 20 1 167837 Coding 1 141gtgcttcatgtaaccctgca 88 21 1 167846 Coding 3 1369 tggcctgctgggccacctgg66 22 1 167848 Coding 3 3278 tgctccagtagtctttcagg 81 23 1 167851 Coding3 3326 tgacctcagggtccaaatcc 41 24 1 304739 5′UTR 3 401ctctagggatgaactgagca 62 25 1 304740 5′UTR 3 408 cagctgcctctagggatgaa 4426 1 304741 5′UTR 1 17 ttcctggagcagctgcctct 57 27 1 304742 5′UTR 1 24acctctgttcctggagcagc 78 28 1 304743 Start Codon 1 29atggcacctctgttcctgga 78 29 1 304744 Start Codon 3 1065gggctgcatggcacctctgt 73 30 1 304745 Coding 3 1086 ggcaacaacaaggagtaccc90 31 1 304746 Coding 3 1090 ggagggcaacaacaaggagt 80 32 1 304747 Coding1 87 agctcgggcagaggccagga 49 33 1 304748 Coding 1 92tctgaagctcgggcagaggc 72 34 1 304749 Coding 1 97 cggcctctgaagctcgggca 1135 1 304750 Coding 3 1267 catcctcggcctctgaagct 49 36 1 304751 Coding 31273 gggaggcatcctcggcctct 65 37 1 304752 Coding 3 1278gagaagggaggcatcctcgg 82 38 1 304753 Coding 3 1281 gctgagaagggaggcatcct75 39 1 304754 Coding 3 1289 tgcatgaagctgagaaggga 74 40 1 304755 Coding1 143 gcgtgcttcatgtaaccctg 95 41 1 304756 Coding 3 1313ttggtggcgtgcttcatgta 92 42 1 304757 Coding 3 1328 gcatccttggcggtcttggt98 43 1 304758 Coding 3 1334 ctcagtgcatccttggcggt 97 44 1 304759 Coding3 1336 tgctcagtgcatccttggcg 93 45 1 304760 Coding 3 1347ctcctgcacgctgctcagtg 65 46 1 304761 Coding 3 1349 gactcctgcacgctgctcag77 47 1 304762 Coding 3 1358 gccacctgggactcctgcac 89 48 1 304763 Coding1 210 gcccctggcctgctgggcca 71 49 1 304764 Coding 1 211agcccctggcctgctgggcc 62 50 1 304765 Coding 3 3253 gaagccatcggtcacccagc71 51 1 304766 Coding 3 3255 ctgaagccatcggtcaccca 85 52 1 304767 Coding3 3265 tttcagggaactgaagccat 73 53 1 304768 Coding 3 3273cagtagtctttcagggaact 40 54 1 304769 Coding 3 3283 aacggtgctccagtagtctt66 55 1 304770 Coding 3 3287 ccttaacggtgctccagtag 88 56 1 304771 Coding3 3295 gaacttgtccttaacggtgc 59 57 1 304772 Coding 3 3301ctcagagaacttgtccttaa 88 58 1 304773 Coding 3 3305 agaactcagagaacttgtcc75 59 1 304774 Coding 3 3310 atcccagaactcagagaact 0 60 1 304775 Coding 33320 cagggtccaaatcccagaac 70 61 1 304776 Coding 3 3332ttggtctgacctcagggtcc 90 62 1 304777 Coding 3 3333 gttggtctgacctcagggtc84 63 1 304778 Coding 3 3339 gctgaagttggtctgacctc 81 64 1 304779 Coding3 3347 cagccacggctgaagttggt 75 65 1 304780 Stop Codon 3 3351caggcagccacggctgaagt 83 66 1 304781 Stop Codon 3 3361attgaggtctcaggcagcca 79 67 1 304782 3′UTR 3 3385 tggataggcaggtggacttg 6468 1 304783 3′UTR 1 369 ctcgcaggatggataggcag 76 69 1 304784 3′UTR 1 374aggagctcgcaggatggata 58 70 1 304785 3′UTR 1 380 gacccaaggagctcgcagga 7371 1 304786 3′UTR 1 385 tgcaggacccaaggagctcg 92 72 1 304787 3′UTR 3 3417tggagattgcaggacccaag 88 73 1 304788 3′UTR 3 3422 agccctggagattgcaggac 6974 1 304789 3′UTR 3 3425 ggcagccctggagattgcag 76 75 1 304790 3′UTR 33445 ccttttaagcaacctacagg 65 76 1 304791 3′UTR 3 3450ctgtcccttttaagcaacct 53 77 1 304792 3′UTR 3 3456 agaatactgtcccttttaag 7278 1 304793 3′UTR 3 3461 cactgagaatactgtccctt 67 79 1 304794 3′UTR 33469 taggagagcactgagaatac 59 80 1 304795 3′UTR 3 3472gggtaggagagcactgagaa 74 81 1 304796 3′UTR 3 3509 aggccagcatgcctggaggg 6382 1 304797 3′UTR 3 3514 ttgggaggccagcatgcctg 55 83 1 304798 3′UTR 33521 agctttattgggaggccagc 90 84 1 304799 3′UTR 3 3526tgtccagctttattgggagg 85 85 1 304800 3′UTR 3 3528 cttgtccagctttattggga 9486 1 304801 3′UTR 3 3533 agcttcttgtccagctttat 74 87 1 304802 3′UTR 33539 catagcagcttcttgtccag 73 88 1 304803 exon:intron 3 416acctggagcagctgcctcta 87 89 1 junction 304804 exon:intron 3 424agggcattacctggagcagc 68 90 1 junction 304805 intron:exon 3 1053acctctgttcctgcaaggaa 74 91 1 junction 304806 exon:intron 3 1121aagtgcttacgggcagaggc 78 92 1 junction 304807 exon:intron 3 1380gcgggtgtacctggcctgct 52 93 1 junction 304808 intron 3 2337aaccctgttgtgaactgcac 59 94 1 304809 intron 3 2405 agtgagcaataccgcctgag80 95 1 304810 intron 3 2542 cgggcttgaattaggtcagg 56 96 1

As shown in the table above, SEQ ID NOs 19, 20, 21, 22, 23, 25, 27, 28,29, 30, 31, 32, 33, 34, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 55, 56, 57, 58, 59, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95 and 96 demonstrated at least 45%inhibition of human apolipoprotein expression in this assay and aretherefore preferred. More preferred are SEQ ID NOs 75, 86 and 85. Thetarget regions to which these preferred sequences are complementary areherein referred to as “preferred target segments” and are thereforepreferred for targeting by compounds of the present invention. Thesepreferred target segments are shown in a Table 123 below. The sequencesrepresent the reverse complement of the preferred antisense compoundsshown in the table above. “Target site” indicates the first (5′-most)nucleotide number on the particular target nucleic acid to which theoligonucleotide binds. Also shown in Table 123 is the species in whicheach of the preferred target segments was found.

Example 115: Antisense Inhibition of Mouse Apolipoprotein Expression byChimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and aDeoxy Gap

In accordance with the present invention, a second series of antisensecompounds was designed to target different regions of the mouseapolipoprotein RNA, using published sequences (GenBank accession numberL04150.1, incorporated herein as SEQ ID NO: 11). The compounds are shownin Table 2. “Target site” indicates the first (5′-most) nucleotidenumber on the particular target nucleic acid to which the compoundbinds. All compounds in Table 2 are chimeric oligonucleotides(“gapmers”) 20 nucleotides in length, composed of a central “gap” regionconsisting of ten 2′-deoxynucleotides, which is flanked on both sides(5′ and 3′ directions) by five-nucleotide “wings”. The wings arecomposed of 2′-O-(2-methoxyethyl) nucleotides, also known as (2′-MOE)nucleotides. The internucleoside (backbone) linkages arephosphorothioate (P═S) throughout the oligonucleotide. All cytidineresidues are 5-methylcytidines. The compounds were analyzed for theireffect on mouse apolipoprotein C-III mRNA levels by quantitativereal-time PCR as described in other examples herein. Data are averagesfrom three experiments in which mouse primary hepatocyte cells weretreated with the antisense oligonucleotides of the present invention. Ifpresent, “N. D.” indicates “no data”.

TABLE 122Inhibition of mouse apolipoprotein C-III mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET TARGET % SEQ ID ISIS # REGION SEQ ID NO SITE SEQUENCE INHIB NO167858 5′UTR 11 1 tagggataaaactgagcagg 47 97 167859 5′UTR 11 21ctggagtagctagctgcttc 30 98 167860 start codon 11 41 gctgcatggcacctacgtac80 99 167861 coding 11 62 ccacagtgaggagcgtccgg 86 100 167862 coding 1188 ggcagatgccaggagagcca 55 101 167863 coding 11 104 ctacctcttcagctcgggca56 102 167864 coding 11 121 cagcagcaaggatccctcta 83 103 167865 coding 11131 gcacagagcccagcagcaag 49 104 167867 coding 11 215ccctggccaccgcagctata 67 105 167868 coding 11 239 atctgaagtgattgtccatc 11106 167869 coding 11 254 agtagcctttcaggaatctg 57 107 167870 coding 11274 cttgtcagtaaacttgctcc 89 108 167871 coding 11 286gaagccggtgaacttgtcag 55 109 167872 coding 11 294 gaatcccagaagccggtgaa 29110 167873 coding 11 299 ggttagaatcccagaagccg 55 111 167874 coding 11319 tggagttggttggtcctcag 79 112 167875 stop codon 11 334tcacgactcaatagctggag 77 113 167877 3′UTR 11 421 cccttaaagcaaccttcagg 71114 167878 3′UTR 11 441 agacatgagaacatactttc 81 115 167879 3′UTR 11 471catgtttaggtgagatctag 87 116 167880 3′UTR 11 496 tcttatccagctttattagg 98117

As shown in the table above, SEQ ID NOs 97, 99, 100, 101, 102, 103, 104,105, 107, 108, 109, 111, 112, 113, 114, 115, 116 and 117 demonstrated atleast 45% inhibition of mouse apolipoprotein C-III expression in thisexperiment and are therefore preferred. More preferred are SEQ ID NOs117, 116, and 100. The target regions to which these preferred sequencesare complementary are herein referred to as “preferred target segments”and are therefore preferred for targeting by compounds of the presentinvention. These preferred target segments are shown in the table below.The sequences represent the reverse complement of the preferredantisense compounds shown in the table above. These sequences are shownto contain thymine (T) but one of skill in the art will appreciate thatthymine (T) is generally replaced by uracil (U) in RNA sequences.“Target site” indicates the first (5′-most) nucleotide number on theparticular target nucleic acid to which the oligonucleotide binds. Alsoshown in Table 3 is the species in which each of the preferred targetsegments was found.

TABLE 123 Sequence and position of preferred target segments identifiedin apolipoprotein C-III. TARGET SITE SEQ TARGET REV COMP SEQ ID ID ID NOSITE SEQUENCE OF SEQ ID ACTIVE IN NO 82975 3 414 cctagaggcagctgctccag 19H. sapiens 118 82980 3 1292 cttctcagcttcatgcaggg 20 H. sapiens 119 829811 141 tgcagggttacatgaagcac 21 H. sapiens 120 82985 3 1369ccaggtggcccagcaggcca 22 H. sapiens 121 82987 3 3278 cctgaaagactactggagca23 H. sapiens 122 220510 3 401 tgctcagttcatccctagag 25 H. sapiens 123220512 1 17 agaggcagctgctccaggaa 27 H. sapiens 124 220513 1 24gctgctccaggaacagaggt 28 H. sapiens 125 220514 1 29 tccaggaacagaggtgccat29 H. sapiens 126 220515 3 1065 acagaggtgccatgcagccc 30 H. sapiens 127220516 3 1086 gggtactccttgttgttgcc 31 H. sapiens 128 220517 3 1090actccttgttgttgccctcc 32 H. sapiens 129 220518 1 87 tcctggcctctgcccgagct33 H. sapiens 130 220519 1 92 gcctctgcccgagcttcaga 34 H. sapiens 131220521 3 1267 agcttcagaggccgaggatg 36 H. sapiens 132 220522 3 1273agaggccgaggatgcctccc 37 H. sapiens 133 220523 3 1278ccgaggatgcctcccttctc 38 H. sapiens 134 220524 3 1281aggatgcctcccttctcagc 39 H. sapiens 135 220525 3 1289tcccttctcagcttcatgca 40 H. sapiens 136 220526 1 143 cagggttacatgaagcacgc41 H. sapiens 137 220527 3 1313 tacatgaagcacgccaccaa 42 H. sapiens 138220528 3 1328 accaagaccgccaaggatgc 43 H. sapiens 139 220529 3 1334accgccaaggatgcactgag 44 H. sapiens 140 220530 3 1336cgccaaggatgcactgagca 45 H. sapiens 141 220531 3 1347cactgagcagcgtgcaggag 46 H. sapiens 142 220532 3 1349ctgagcagcgtgcaggagtc 47 H. sapiens 143 220533 3 1358gtgcaggagtcccaggtggc 48 H. sapiens 144 220534 1 210 tggcccagcaggccaggggc49 H. sapiens 145 220535 1 211 ggcccagcaggccaggggct 50 H. sapiens 146220536 3 3253 gctgggtgaccgatggcttc 51 H. sapiens 147 220537 3 3255tgggtgaccgatggcttcag 52 H. sapiens 148 220538 3 3265atggcttcagttccctgaaa 53 H. sapiens 149 220540 3 3283aagactactggagcaccgtt 55 H. sapiens 150 220541 3 3287ctactggagcaccgttaagg 56 H. sapiens 151 220542 3 3295gcaccgttaaggacaagttc 57 H. sapiens 152 220543 3 3301ttaaggacaagttctctgag 58 H. sapiens 153 220544 3 3305ggacaagttctctgagttct 59 H. sapiens 154 220546 3 3320gttctgggatttggaccctg 61 H. sapiens 155 220547 3 3332ggaccctgaggtcagaccaa 62 H. sapiens 156 220548 3 3333gaccctgaggtcagaccaac 63 H. sapiens 157 220549 3 3339gaggtcagaccaacttcagc 64 H. sapiens 158 220550 3 3347accaacttcagccgtggctg 65 H. sapiens 159 220551 3 3351acttcagccgtggctgcctg 66 H. sapiens 160 220552 3 3361tggctgcctgagacctcaat 67 H. sapiens 161 220553 3 3385caagtccacctgcctatcca 68 H. sapiens 162 220554 1 369 ctgcctatccatcctgcgag69 H. sapiens 163 220555 1 374 tatccatcctgcgagctcct 70 H. sapiens 164220556 1 380 tcctgcgagctccttgggtc 71 H. sapiens 165 220557 1 385cgagctccttgggtcctgca 72 H. sapiens 166 220558 3 3417cttgggtcctgcaatctcca 73 H. sapiens 167 220559 3 3422gtcctgcaatctccagggct 74 H. sapiens 168 220560 3 3425ctgcaatctccagggctgcc 75 H. sapiens 169 220561 3 3445cctgtaggttgcttaaaagg 76 H. sapiens 170 220562 3 3450aggttgcttaaaagggacag 77 H. sapiens 171 220563 3 3456cttaaaagggacagtattct 78 H. sapiens 172 220564 3 3461aagggacagtattctcagtg 79 H. sapiens 173 220565 3 3469gtattctcagtgctctccta 80 H. sapiens 174 220566 3 3472ttctcagtgctctcctaccc 81 H. sapiens 175 220567 3 3509ccctccaggcatgctggcct 82 H. sapiens 176 220568 3 3514caggcatgctggcctcccaa 83 H. sapiens 177 220569 3 3521gctggcctcccaataaagct 84 H. sapiens 178 220570 3 3526cctcccaataaagctggaca 85 H. sapiens 179 220571 3 3528tcccaataaagctggacaag 86 H. sapiens 180 220572 3 3533ataaagctggacaagaagct 87 H. sapiens 181 220573 3 3539ctggacaagaagctgctatg 88 H. sapiens 182 220574 3 416 tagaggcagctgctccaggt89 H. sapiens 183 220575 3 424 gctgctccaggtaatgccct 90 H. sapiens 184220576 3 1053 ttccttgcaggaacagaggt 91 H. sapiens 185 220577 3 1121gcctctgcccgtaagcactt 92 H. sapiens 186 220578 3 1380agcaggccaggtacacccgc 93 H. sapiens 187 220579 3 2337gtgcagttcacaacagggtt 94 H. sapiens 188 220580 3 2405ctcaggcggtattgctcact 95 H. sapiens 189 220581 3 2542cctgacctaattcaagcccg 96 H. sapiens 190 82997 11 1 cctgctcagttttatcccta97 M. musculus 191 82999 11 41 gtacgtaggtgccatgcagc 99 M. musculus 19283000 11 62 ccggacgctcctcactgtgg 100 M. musculus 193 83001 11 88tggctctcctggcatctgcc 101 M. musculus 194 83002 11 104tgcccgagctgaagaggtag 102 M. musculus 195 83003 11 121tagagggatccttgctgctg 103 M. musculus 196 83004 11 131cttgctgctgggctctgtgc 104 M. musculus 197 83006 11 215tatagctgcggtggccaggg 105 M. musculus 198 83008 11 254cagattcctgaaaggctact 107 M. musculus 199 83009 11 274ggagcaagtttactgacaag 108 M. musculus 200 83010 11 286ctgacaagttcaccggcttc 109 M. musculus 201 83012 11 299cggcttctgggattctaacc 111 M. musculus 202 83013 11 319ctgaggaccaaccaactcca 112 M. musculus 203 83014 11 334ctccagctattgagtcgtga 113 M. musculus 204 83016 11 421cctgaaggttgctttaaggg 114 M. musculus 205 83017 11 441gaaagtatgttctcatgtct 115 M. musculus 206 83018 11 471ctagatctcacctaaacatg 116 M. musculus 207 83019 11 496cctaataaagctggataaga 117 M. musculus 208

As these “preferred target segments” have been found by experimentationto be open to, and accessible for, hybridization with the antisensecompounds of the present invention, one of skill in the art willrecognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother compounds that specifically hybridize to these preferred targetsegments and consequently inhibit the expression of apolipoprotein

According to the present invention, antisense compounds includeantisense oligomeric compounds, antisense oligonucleotides, ribozymes,external guide sequence (EGS) oligonucleotides, alternate splicers,primers, probes, and other short oligomeric compounds that hybridize toat least a portion of the target nucleic acid.

Example 116: Antisense Inhibition of Human Apolipoprotein C-IIIExpression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOEWings and a Deoxy Gap—Additional Antisense Compounds

In accordance with the present invention, an additional series ofantisense compounds was designed to target different regions of thehuman apolipoprotein C-III RNA, using published sequences (nucleotides6238608 to 6242565 of the sequence with GenBank accession numberNT_035088.1, representing a genomic sequence, incorporated herein as SEQID NO: 3, and GenBank accession number NM_000040.1, incorporated hereinas SEQ ID NO: 1). The compounds are shown in Table 124. “Target site”indicates the first (5′-most) nucleotide number on the particular targetsequence to which the compound binds. All compounds in Table 124 arechimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composedof a central “gap” region consisting of ten 2′-deoxynucleotides, whichis flanked on both sides (5′ and 3′ directions) by five-nucleotide“wings”. The wings are composed of 2′-O-(2-methoxyethyl) nucleotides,also known as (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on human apolipoprotein C-III mRNA levels by quantitativereal-time PCR as described in other examples herein. Data are averagesfrom three experiments in which HepG2 cells were treated with theantisense oligonucleotides of the present invention. If present, “N. D.”indicates “no data”.

TABLE 124 Inhibition of human apolipoprotein C-III mRNAlevels by chimeric phosphorothioateoligonucleotides having 2′-MOE wings and a deoxy gap TARGET SEQ SEQ IDTARGET % ID ISIS # NO SITE SEQUENCE INHIB NO 167826 3 1063gctgcatggcacctctgttc 0 209 167828 3 1110 ggcagaggccaggagcgcca 0 210167830 1 91 ctgaagctcgggcagaggcc 9 211 167832 1 101 tcctcggcctctgaagctcg0 212 167840 3 1315 tcttggtggcgtgcttcatg 0 213 167842 3 1335gctcagtgcatccttggcgg 38 214 167844 3 1345 cctgcacgctgctcagtgca 28 215167847 3 3256 actgaagccatcggtcaccc 0 216 167850 3 3306cagaactcagagaacttgtc 0 217 167852 3 3336 gaagttggtctgacctcagg 0 218167853 3 3420 ccctggagattgcaggaccc 0 219 167854 3 3426gggcagccctggagattgca 22 220 167855 3 3446 cccttttaagcaacctacag 27 221

Example 117: Antisense Inhibition of Human Apolipoprotein C-IIIExpression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOEWings and a Deoxy Gap: Dose-Response Study in HepG2 Cells

In accordance with the present invention, a subset of the antisenseoligonucleotides from Examples 15 and 17 was further investigated in adose-response study. Treatment doses of ISIS 167842 (SEQ ID NO: 214),ISIS 167844 (SEQ ID NO: 215), ISIS 167846 (SEQ ID NO: 22), ISIS 167837(SEQ ID NO: 21), ISIS 304789 (SEQ ID NO: 75), ISIS 304799 (SEQ ID NO:85), and ISIS 304800 (SEQ ID: 86) were 50, 150 and 300 nM. The compoundswere analyzed for their effect on human apolipoprotein C-III mRNA levelsin HepG2 cells by quantitative real-time PCR as described in otherexamples herein. Data are averages from two experiments and are shown inthe table below.

TABLE 125 Inhibition of human apolipoprotein C-III mRNA levels bychimeric phosphorothioate oligonucleotides having 2′-MOE wings and adeoxy gap Dose of oligonucleotide 50 nM 150 nM 300 nM ISIS # SEQ ID NOPercent Inhibition 167842 214 88 77 92 167844 215 86 86 84 167846 22 7980 79 167837 21 83 86 84 304789 75 81 91 92 304799 85 82 93 88 304800 8680 86 91

These data demonstrate that the expression of apolipoprotein C-III isinhibited in a dose-dependent manner upon treatment of cells withantisense compounds targeting apolipoprotein C-III. These compounds werefurther analyzed in Hep3B cells for their ability to reduce mRNA levelsin Hep3B cells and it was determined that ISIS 167842 and 167837inhibited apolipoprotein C-III expression in a dose dependent manner inthis cell line as well.

Example 118: Antisense Inhibition of Apolipoprotein C-III in CynomolgusMonkey Primary Hepatocytes

In a further embodiment, antisense compounds targeted to humanapolipoprotein C-III were tested for their effects on apolipoproteinC-III expression in primary Cynomolgus monkey hepatocytes. Pre-platedprimary Cynomolgus monkey hepatocytes were purchased from InVitroTechnologies (Baltimore, Md.). Cells were cultured in high-glucose DMEM(Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10%fetal bovine serum (Invitrogen Life Technologies, Carlsbad, Calif.), 100units/mL and 100 μg/mL streptomycin (Invitrogen Life Technologies,Carlsbad, Calif.).

Cells at a density of 80,000 cells per well in a 24-well plate weretreated with 10, 50, 150 and 300 nM of ISIS 304789 (SEQ ID NO: 75), ISIS304799 (SEQ ID NO: 85) or ISIS 304800 (SEQ ID NO: 86). ISIS 113529(CTCTTACTGTGCTGTGGACA, SEQ ID NO: 17) served as a controloligonucleotide. ISIS 113529 is a chimeric oligonucleotide (“gapmer”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-O-(2-methoxyethyl) nucleotides, also known as (2′-MOE) nucleotides.The internucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines.

Following 24 hours of treatment with antisense oligonucleotides,apolipoprotein C-III mRNA was measured by real-time PCR as described byother examples herein, using the primers and probe designed to the humanapolipoprotein C-III sequence (forward primer: TCAGCTTCATGCAGGGTTACAT(SEQ ID NO: 5) reverse primer: ACGCTGCTCAGTGCATCCT (SEQ ID NO: 6) andthe PCR probe was: FAM-AAGCACGCCACCAAGACCGCC-TAMRA (SEQ ID NO: 7)) tomeasure Cynomolgous monkey apolipoprotein C-III mRNA. Primers and probedesigned to human GAPDH (forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO:8) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 9) and the PCR probewas: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10)) were used tomeasure Cynomolgous monkey GAPDH mRNA expression, for the purpose ofnormalizing gene target quantities obtained by real-time PCR. Untreatedcells served as the control to which data were normalized. Data are theaverage of three experiments and are presented in the table below.

TABLE 126 Antisense inhibition of apolipoprotein C-III in Cynomolgusmonkey primary hepatocytes Dose of Oligonucleotide 10 nM 50 nM 150 nM300 nM ISIS # SEQ ID NO % Inhibition 304789 75 0 7 1 55 304799 85 34 6066 48 304800 86 9 53 59 57 113529 222 N.D. N.D. 0 0

Example 119: Cynomolgus Monkey Apolipoprotein C-III Sequence

In a further embodiment, a portion of the Cynomolgus monkeyapolipoprotein C-III gene was sequenced. Positions 8 to 476 of the humanapolipoprotein C-III mRNA sequence (incorporated in its entirety hereinas SEQ ID NO: 1) contain the target segment to which ISIS 304789hybridizes. The corresponding region of Cynomolgus monkey apolipoproteinC-III mRNA was sequenced. RNA was isolated and purified from primaryCynomolgus monkey hepatocytes (InVitro Technologies, Gaithersburg, Md.)and was subjected to a reverse transcriptase reaction (kit fromInvitrogen Life Technologies, Carlsbad, Calif.). The resultant cDNA wasthe substrate for 40 rounds of PCR amplification, using 5′ and 3′primers designed to positions 8 and 476, respectively, of the humanapolipoprotein C-III mRNA (Amplitaq PCR kit, Invitrogen LifeTechnologies, Carlsbad, Calif.). Following gel purification of theresultant 468 bp fragment, the forward and reverse sequencing reactionsof each product were performed by Retrogen (San Diego, Calif.). ThisCynomolgus monkey sequence is incorporated herein as SEQ ID NO: 223 andis 92% identical to positions 8 to 476 of the human apolipoprotein C-IIImRNA.

Example 120: Chimeric Phosphorothioate Oligonucleotide Having 2′-MOEWings and a Deoxy Gap, Targeted to Cynomolgus Monkey ApolipoproteinC-III

In a further embodiment, the sequence of Cynomolgus monkeyapolipoprotein C-III incorporated herein as SEQ ID NO: 223 was used todesign an antisense oligonucleotide having 100% complementarity toCynomolgus apolipoprotein C-III mRNA. ISIS 340340 (GGCAGCCCTGGAGGCTGCAG;incorporated herein as SEQ ID NO: 18) targets nucleotide 397 of SEQ IDNO: 223, within a region corresponding to the 3′ UTR of the humanapolipoprotein C-III to which ISIS 304789 hybridizes. ISIS 340340 is achimeric oligonucleotide (“gapmer”) 20 nucleotide in length composed ofa central “gap” region consisting of 2′deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by 5 nucleotide “wings”.The wings are composed of 2′methoxyethyl (2′-MOE) nucleotides.Internucleoside (backbone) linkages are phosphorothioate (P═S)throughout the nucleotide. All cytidine residues are 5-methyl cytidines.

1.-25. (canceled)
 26. A compound comprising a single stranded modifiedoligonucleotide and a conjugate group, wherein the modifiedoligonucleotide consists of 12 to 30 linked nucleosides and comprises anucleobase sequence comprising a portion of at least 8 contiguousnucleobases complementary to an equal length portion of nucleobases 3533to 3552 of SEQ ID NO: 3, wherein the nucleobase sequence of the modifiedoligonucleotide is at least 80% complementary to SEQ ID NO: 3; whereinthe conjugate group is linked to the modified oligonucleotide at the 5′end of the modified oligonucleotide by a conjugate linker; and whereinthe conjugate group comprises:

wherein each n is, independently, from 1 to
 20. 27. The compound ofclaim 26, wherein the nucleobase sequence of the modifiedoligonucleotide is at least 85%, at least 90%, at least 95%, or 100%complementary to SEQ ID NOs:
 3. 28. The compound of claim 26, whereinthe modified oligonucleotide comprises at least one modifiedinternucleoside linkage.
 29. The compound of claim 28, wherein themodified internucleoside linkage is a phosphorothioate internucleosidelinkage.
 30. The compound of claim 26, wherein the modifiedoligonucleotide comprises at least one modified sugar.
 31. The compoundof claim 30, wherein at least one modified sugar is selected from abicyclic sugar, a 2′-O-methoxyethyl modified sugar, a constrained ethylmodified sugar, a 3′-fluoro-HNA or a 4′-(CH₂)_(n)—O-2′ bridge, wherein nis 1 or
 2. 32. The compound of claim 26, wherein at least one nucleosidecomprises a modified nucleobase.
 33. The compound of claim 32, whereinthe modified nucleobase is a 5-methylcytosine.
 34. The compound of claim26, wherein the modified oligonucleotide comprises: a gap segmentconsisting of linked deoxynucleosides; a 5′ wing segment consisting oflinked nucleosides; a 3′ wing segment consisting of linked nucleosides;wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment and wherein each nucleoside of each wing segmentcomprises a modified sugar.
 35. The compound claim 26, wherein themodified oligonucleotide comprises: a gap segment consisting of tenlinked deoxynucleosides; a 5′ wing segment consisting of five linkednucleosides; a 3′ wing segment consisting of five linked nucleosides;wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a 2′-O-methoxyethyl sugar, and wherein each cytosine residueis a 5-methylcytosine.
 36. The compound of claim 26, wherein eachinternucleoside linkage of the modified oligonucleotide is aphosphorothioate linkage.
 37. The compound of claim 35, wherein themodified oligonucleotide consists of 20 linked nucleosides.
 38. Thecompound of claim 26, wherein the conjugate linker has a structureselected from among:

wherein each L is, independently, a phosphorus linking group or aneutral linking group; and each n is, independently, from 1 to
 20. 39.The compound of claim 26, wherein the conjugate linker has a structureselected from among:


40. The compound of claim 26, wherein the conjugate linker has thefollowing structure:


41. The compound of claim 26, wherein the conjugate linker has thefollowing structure:


42. The compound of claim 26, wherein the conjugate linker has astructure selected from among:


43. The compound of claim 26, wherein the conjugate linker has astructure selected from among:


44. The compound of claim 26, wherein the conjugate linker has astructure selected from among:


45. The compound of claim 26, wherein the conjugate linker has astructure selected from among:

wherein each n is, independently, is from 1 to 20; and p is from 1 to 6.46. The compound of claim 26, wherein the conjugate linker has astructure selected from among:

wherein each n is, independently, from 1 to
 20. 47. The compound ofclaim 26, wherein the conjugate linker has a structure selected fromamong:


48. The compound of claim 26, wherein the conjugate linker has astructure selected from among:

wherein n is from 1 to
 20. 49. The compound of claim 26, wherein theconjugate linker has a structure selected from among:


50. The compound of claim 26, wherein the conjugate linker has astructure selected from among:

wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or
 7. 51. Thecompound of claim 26, wherein the conjugate linker has the followingstructure:


52. The compound of claim 26, wherein the conjugate group comprises:


53. The compound of claim 26, wherein the conjugate group comprises:

and the conjugate linker has the following structure:


54. The compound of claim 26, wherein the conjugate group comprises acleavable moiety selected from a phosphodiester, an amide, or an ester.55. The compound of claim 26, wherein the conjugate comprises:


56. A pharmaceutical composition comprising the compound of claim 26 anda pharmaceutically acceptable carrier or diluent.
 57. A methodcomprising administering to an animal the composition of claim
 56. 58.The method of claim 57 wherein the animal is a human.
 59. The method ofclaim 57, for use in treating, preventing, or slowing progression of adisease related to elevated ApoCIII.
 60. The method of claim 57, whereinadministering the compound prevents, treats, ameliorates, or slowsprogression of a cardiovascular, metabolic and/or inflammatory disease,disorder or condition in the animal.
 61. The method of claim 57, whereinthe animal has, or is at risk of having, any one or more ofhypertriglyceridemia, Fredrickson Type I dyslipidemia, FCS, LPLD,pancreatitis, diabetes, insulin insensitivity.